Molecular characterization of Urosporidium tapetis sp. nov., a haplosporidian hyperparasite infecting metacercariae of Parvatrema duboisi (Dollfus 1923), a trematode parasite of Manila clam Ruditapes philippinarum on the west coast of Korea.

Recently, a putative new hyperparasitic haplosporidian in the genus Urosporidium was identified from metacercariae of the trematode Parvatrema duboisi infecting Manila clam Ruditapes philippinarum on the west coast of Korea. In this study, we applied small subunit ribosomal DNA (SSU rDNA) sequences as a marker to substantiate the phylogenetic relationship of the unidentified Urosporidium within the Order Haplosporida. In our phylogenetic analysis, the 1890 bp of SSU rDNA sequences obtained were closely related to a haplosporidian parasite forming a sister clade to Urosporidium group, although the gene sequences were only 89.22-89.70% similar to Urosporidium spp. Such molecular phylogenetic distance within the genus suggested that the unidentified Urosporidium is a new member of the genus. Accordingly, we report the unidentified haplosporidian hyperparasite as Urosporidium tapetis sp. nov.

Isolation and Molecular Analysis of a Novel Neorickettsia Species That Causes Potomac Horse Fever.

Potomac horse fever (PHF), a severe and frequently fatal febrile diarrheal disease, has been known to be caused only by Neorickettsia risticii, an endosymbiont of digenean trematodes. Here, we report the cell culture isolation of a new Neorickettsia species found in two locations in eastern Ontario, Canada, in 2016 and 2017 (in addition to 10 variable strains of N. risticii) from N. risticii PCR-negative horses with clinical signs of PHF. Gene sequences of 16S rRNA and the major surface antigen P51 of this new Neorickettsia species were distinct from those of all previously characterized N. risticii strains and Neorickettsia species, except for those from an uncharacterized Neorickettsia species culture isolate from a horse with PHF in northern Ohio in 1991. The new Neorickettsia species nonetheless had the characteristic intramolecular repeats within strain-specific antigen 3 (Ssa3), which were found in all sequenced Ssa3s of N. risticii strains. Experimental inoculation of two naive ponies with the new Neorickettsia species produced severe and subclinical PHF, respectively, and the bacteria were reisolated from both of them, fulfilling Koch's postulates. Serological assay titers against the new Neorickettsia species were higher than those against N. risticii Whole-genome sequence analysis of the new Neorickettsia species revealed unique features of this bacterium compared with N. risticii We propose to classify this new bacterium as Neorickettsia finleia sp. nov. This finding will improve the laboratory diagnosis of and vaccine for PHF, environmental risk assessment of PHF, and understanding of PHF pathogenesis and Neorickettsia biology in general.IMPORTANCE Despite the detection of Neorickettsia species DNA sequences in various trematode species and their hosts, only three Neorickettsia species have been cell culture isolated and whole-genome sequenced and are known to infect mammals and/or cause disease. The molecular mechanisms that enable the obligatory intracellular bacterium Neorickettsia to colonize trematodes and to horizontally transmit from trematodes to mammals, as well as the virulence factors associated with specific mammalian hosts, are unknown. Potomac horse fever (PHF) is a severe and acute systemic infectious disease of horses, with clinical signs that include diarrhea. Neorickettsia risticii is the only known bacterial species that causes PHF. Ingestion of insects harboring N. risticii-infected trematodes by horses leads to PHF. Our discovery of a new Neorickettsia species that causes PHF and whole-genome sequence analysis of this bacterium will improve laboratory diagnosis and vaccine development for PHF and will contribute to our understanding of Neorickettsia ecology, pathogenesis, and biology.

Ultrastructure and localization of Neorickettsia in adult digenean trematodes provides novel insights into helminth-endobacteria interaction.

BACKGROUND: Neorickettsia are a group of intracellular α proteobacteria transmitted by digeneans (Platyhelminthes, Trematoda). These endobacteria can also infect vertebrate hosts of the helminths and cause serious diseases in animals and humans. Neorickettsia have been isolated from infected animals and maintained in cell cultures, and their morphology in mammalian cells has been described. However, limited information is available on the morphology and localization of Neorickettsia in the trematode host. METHODS: We used a Neorickettsia-infected strain of the model trematode Plagiorchis elegans to infect Syrian Golden hamsters to produce adult worms. Ultrastructure of Neorickettsia was assessed by transmission electron microscopy of high pressure freezing/freeze substitution fixed specimens. A Neorickettsia surface protein from P. elegans (PeNsp-3) was cloned and antibodies against the recombinant protein were used to localize Neorickettsia by immunohistochemistry. RESULTS: Ultrastructural analysis revealed moderate numbers of pleomorphic endobacteria with a median size of 600 × 400 nm and characteristic double membranes in various tissue types. Endobacteria showed tubular membrane invaginations and secretion of polymorphic vesicles. Endobacteria were unevenly localized as single cells, or less frequently as small morula-like clusters in the ovary, Mehlis' gland, vitelline follicles, uterus, intrauterine eggs, testis, cirrus-sac, tegument, intestine and the oral and ventral sucker. Examination of hamster small intestine infected with P. elegans showed many endobacteria at the host-parasite interface such as the oral and ventral sucker, the tegument and the excretory pore. CONCLUSIONS: We conclude that adult P. elegans trematodes carry Neorickettsia endobacteria in varying numbers in many tissue types that support vertical transmission, trematode to trematode transmission via seminal fluid, and possibly horizontal transmission from trematodes to vertebrate hosts. These means appear to be novel mechanisms of pathogen transmission by endoparasitic worms.

Trematode Aspidogastrea found in the freshwater mussels in the Yangtze River basin / Trematodos Aspidogastrea encontrados en los mejillones de agua dulce en la cuenca del río Yangtze

Objective: To investigate the prevalence of trematode Aspidogastrea in the freshwater mussels in the Yangtze River basin within Anhui province, China. Methods: We initially harvested the freshwater mussels living in the Yangtze River running through Anhui area, and labeled them with corresponding number. Then the samples were dissected for isolating the flukes, which were identified by conventional staining. Results: Infection rate of trematode Aspidogastrea in freshwater mussels in the Yangtze River basin within the territory of Anhui province was 30.38% (103/339) in general, and a total of 912 flukes of Aspidogastrea were detected in the 103 mussels, with average infection rate of 8.85 for each mussel. Conclusion: Trematode Aspidogastrea is prevalent in the freshwater bivalves living in the Yangtze River basin running through Anhui area, and the treamatode was identified as Aspidogaster sp. belong to Aspidogaste under Aspidogastridae of Aspidogastrea (AU)

Objetivo: investigar la prevalencia de trematodos Aspidogastrea en mejillones de agua dulce en la cuenca del río Yangtze en la provincia de Anhui, China. Métodos: se recogieron mejillones de agua dulce en el río Yangtze a su paso por la provincia de Anhui y se etiquetaron con su número correspondiente. Posteriormente se disecaron para aislar los trematodos por medio de tinción convencional. Resultados: la tasa de infección de trematodos en mejillones de agua dulce en la cuenca del río Yangtze, en el territorio de la provincia de Anhui fue 30,38% (103/339), en general, y un total de 912 trematodos fueron detectados en 103 mejillones, con tasa promedio de infección de 8,85 por cada mejillón. Conclusión: el trematodo Aspidogastrea es frecuente en los bivalvos de agua dulce que viven en la cuenca del río Yangtze, en la región de Anhui, y el trematodo fue identifi cado como Aspidogaster sp. pertenecen a la familia Aspidogaste bajo el género Aspidogastridae de Aspidogastrea (AU)

Effect of the Filamentous Fungus Mucor circinelloides On The Development of Eggs of the Rumen Fluke Calicophoron daubneyi (Paramphistomidae).

Ruminants infected by Paramphistomidae flukes shed eggs in the feces, which pass through different stages in the environment until the infective stages (metacercariae) are reached. The activity of the soil fungus Mucor circinelloides on the development of eggs of the rumen fluke Calicophoron daubneyi was presently tested with 3 probes, i.e., in petri plates, feces, and an aqueous environment (tubes). The effect of the fungus was assessed by recording the numbers of undeveloped, nonviable, and embryonated eggs. Nonviable eggs were considered when vacuolization occurred, the inner structures were not clearly observed, the eggshell was broken, or the embryo inside was destroyed. By considering the ability of hyphae of M. circinelloides to develop in the presence of C. daubneyi eggs, attach to their surface, and penetrate and destroy the inner embryo, this ovicidal effect was classified as type 3. After a period of 50 days, the percentage of undeveloped eggs in the feces of infected cattle was 40%; furthermore, 27% of the eggs were nonviable, and 33% were embryonated (1 miracidium inside). The addition of 4 doses of M. circinelloides spores directly onto the feces resulted in 9-31% undeveloped eggs, 38-60% nonviable eggs, and 9-21% embryonated eggs, and no statistical significances were obtained among the different doses. Placing the eggs of C. daubneyi into an aqueous solution containing 107 spores of M. circinelloides/ml for 29 days resulted in 43% undeveloped eggs, 40% nonviable eggs, and 17% embryonated eggs, whereas in the controls, the percentages were 48%, 12%, and 40%, respectively. These data demonstrate the usefulness of the spores of the fungus M. circinelloides in limiting the development of the eggs of the trematode C. daubneyi.

Molecular investigation of Neorickettsia risticii in trematodes and snails in a region with serological evidence of this agent in horses, state of Rio de Janeiro / Pesquisa molecular de Neorickettsia risticii em trematódeos e moluscos em região com evidência sorológica do agente em equinos, estado do Rio de Janeiro

In Brazil, some studies have indicated that Neorickettsia risticii circulates in horses, but it is unclear which are the possible intermediate vectors of this bacterium in the country. The aim of this study was to use molecular techniques in order to analyze the presence of N. risticii in snails and larval stages of trematodes in farms in a region with a history of seroreactive horses towards this bacterium, in Rio de Janeiro, Brazil. Convenience sampling was used in the studied region. The collected snails were exposed to incandescent light (60W) for 2-4 hours in order to investigate trematodes in larval forms. Deoxyribonucleic acid (DNA) was extracted from snail tissue and trematode. Real-time PCR (qPCR) technique was used to investigate the presence of a 16S rRNA gene fragment of N. risticii. Snail specimens (n=410) were collected from 11 horse-breeding farms, and the following species were identified: Melanoides tuberculata, Pomacea sp., Biomphalaria tenagophila, Physa acuta, Drepanotrema anatinum and Biomphalaria straminea. Only 3.17% (n=13/410) of the collected snails were infected by trematodes. The cercariae obtained from these snails were classified as Megalourous cercariae, Pleurolophocercus cercariae and Furcocercous cercariae. There was no amplification of the target DNA of N. risticii in the snail and trematode samples tested by qPCR. Based on these data, the transmission of N. risticii by trematodes using these snail species in this region does not appear to occur or occurs at very low rates. Thus, further studies are needed in order to clarify which species of invertebrate hosts are infected by this bacterium and potentially participate in the transmission chain of equine neorickettsiosis in the state of Rio de Janeiro, Brazil.(AU)

No Brasil, estudos apontam a circulação de Neorickettsia risticii em equinos, contudo não estão claros quais os possíveis vetores intermediários dessa bactéria no país. O objetivo do presente estudo foi analisar a presença de N. risticii, utilizando-se técnicas moleculares, em caramujos e estágios larvais de trematódeos em propriedades rurais de uma região com histórico de equinos sororreativos para essa bactéria, no Rio de Janeiro, Brasil. Uma amostragem por conveniência foi utilizada na região de estudo. Os caramujos coletados foram expostos à luz incandescente (60W) durante duas-quatro horas para a investigação de trematódeos nas formas larvais. A extração de ácido desoxirribonucleico (DNA) foi realizada em tecidos de caramujos e trematódeos. A técnica de PCR em tempo real (qPCR) foi utilizada para investigar a presença de um fragmento do gene 16S rRNA de N. risticii. Foram coletados 410 espécimes de caramujos de 11 propriedades com criações de equinos, sendo identificadas as seguintes espécies: Melanoides tuberculata, Pomacea sp., Biomphalaria tenagophila, Physa acuta, Drepanotrema anatinum e Biomphalaria straminea. Apenas 3,17% (n=13/410) dos caramujos identificados estavam infectados por trematódeos. As cercárias obtidas desses caramujos foram classificadas em Megalourous cercariae, Pleurolophocercus cercariae e Furcocercous cercariae. Não foi observada a amplificação do DNA-alvo de N. risticii, por meio da qPCR, em nenhuma das amostras de caramujos e trematódeos testadas. Com base nesses dados, a transmissão de N. risticii por trematódeos que utilizam as espécies de caramujos nessa região parece não ocorrer ou ocorre a taxas muito reduzidas. Portanto, novos estudos são necessários para elucidar quais espécies de hospedeiros invertebrados se infectam por essa bactéria e potencialmente participam da cadeia de transmissão da neorickettsiose equina no estado do Rio de Janeiro, Brasil.(AU)

Detection of the bacterial endosymbiont Neorickettsia in a New Zealand digenean.

Neorickettsia are endosymbiotic bacteria that infect digeneans (Trematoda). These bacteria are of interest worldwide because of their ability to move from the parasite to its host, where they can cause serious diseases of humans and animals. While several disease-forming species of Neorickettsia have been well studied, and numerous Neorickettsia types have been identified in regions such as North America and parts of Asia, records from other locations are sparse. To date, there have been no reports of Neorickettsia from New Zealand. We screened ten species of digeneans infecting seven native gastropod species (both marine and freshwater) found near Dunedin, New Zealand, for the presence of neorickettsial infections. A >1300 bp long section of 16S rRNA belonging to a Neorickettsia bacterium was isolated from opecoelid digeneans of two individuals of the mudflat topshell snail Diloma subrostrata. These sequences represent the first evidence of neorickettsial infection in native New Zealand animals and are also the first Neorickettsia found in digeneans of the family Opecoelidae.

Anthelmintic Effect of Bacillus thuringiensis Strains against the Gill Fish Trematode Centrocestus formosanus.

Parasitic agents, such as helminths, are the most important biotic factors affecting aquaculture, and the fluke Centrocestus formosanus is considered to be highly pathogenic in various fish species. There have been efforts to control this parasite with chemical helminthicides, but these efforts have had unsuccessful results. We evaluated the anthelmintic effect of 37 strains of Bacillus thuringiensis against C. formosanus metacercariae in vitro using two concentrations of total protein, and only six strains produced high mortality. The virulence (CL50) on matacercariae of three strains was obtained: the GP308, GP526, and ME1 strains exhibited a LC50 of 146.2 µg/mL, 289.2 µg/mL, and 1721.9 µg/mL, respectively. Additionally, these six B. thuringiensis strains were evaluated against the cercariae of C. formosanus; the LC50 obtained from the GP526 strain with solubilized protein was 83.8 µg/mL, and it could be considered as an alternative control of the metacercariae and cercariae of this parasite in the productivity systems of ornamental fishes.

The numbers game: quantitative analysis of Neorickettsia sp. propagation through complex life cycle of its digenean host using real-time qPCR.

Bacteria of the genus Neorickettsia are obligate intracellular endosymbionts of parasitic flukes (Digenea) and are passed through the entire complex life cycle of the parasite by vertical transmission. Several species of Neorickettsia are known to cause diseases in domestic animals, wildlife, and humans. Quantitative data on the transmission of the bacteria through the digenean life cycle is almost completely lacking. This study quantified for the first time the abundance of Neorickettsia within multiple stages of the life cycle of the digenean Plagiorchis elegans. Snails Lymnaea stagnalis collected from a pond in North Dakota were screened for the presence of digenean cercariae, which were subsequently tested for the presence of Neorickettsia. Three L. stagnalis were found shedding P. elegans cercariae infected with Neorickettsia. These snails were used to initiate three separate laboratory life cycles and obtain all life cycle stages for bacterial quantification. A quantitative real-time PCR assay targeting the GroEL gene was developed to enumerate Neorickettsia sp. within different stages of the digenean life cycle. The number of bacteria significantly increased throughout all stages, from eggs to adults. The two largest increases in number of bacteria occurred during the period from eggs to cercariae and from 6-day metacercariae to 48-h juvenile worms. These two periods seem to be the most important for Neorickettsia propagation through the complex digenean life cycle and maturation in the definitive host.

Germs within Worms: Localization of Neorickettsia sp. within Life Cycle Stages of the Digenean Plagiorchis elegans.

Neorickettsia spp. are bacterial endosymbionts of parasitic flukes (Digenea) that also have the potential to infect and cause disease (e.g., Sennetsu fever) in the vertebrate hosts of the fluke. One of the largest gaps in our knowledge of Neorickettsia biology is the very limited information available regarding the localization of the bacterial endosymbiont within its digenean host. In this study, we used indirect immunofluorescence microscopy to visualize Neorickettsia sp. within several life cycle stages of the digenean Plagiorchis elegans Individual sporocysts, cercariae, metacercariae, and adults of P. elegans naturally infected with Neorickettsia sp. were obtained from our laboratory-maintained life cycle, embedded, sectioned, and prepared for indirect immunofluorescence microscopy using anti-Neorickettsia risticiihorse serum as the primary antibody. Neorickettsiasp. was found within the tegument of sporocysts, throughout cercarial embryos (germ balls) and fully formed cercariae (within the sporocysts), throughout metacercariae, and within the tegument, parenchyma, vitellaria, uteri, testes, cirrus sacs, and eggs of adults. Interestingly, Neorickettsia sp. was not found within the ovarian tissue. This suggests that vertical transmission of Neorickettsia within adult digeneans occurs via the incorporation of infected vitelline cells into the egg rather than direct infection of the ooplasm of the oocyte, as has been described for other bacterial endosymbionts of invertebrates (e.g.,Rickettsia and Wolbachia).

Laboratory maintenance of the bacterial endosymbiont, Neorickettsia sp., through the life cycle of a digenean, Plagiorchis elegans.

The Digenea (Platyhelminthes: Trematoda) are a diverse and complex group of internal metazoan parasites. These parasites can serve as hosts to obligate intracellular bacteria belonging to the genus Neorickettsia (Family: Anaplasmataceae). Neorickettsiae persist within all stages of the fluke life cycle and thus are maintained through vertical transmission. However, the low prevalence of Neorickettsia in nature limits study of their transmission biology at different steps of digenean life cycles. To resolve this dilemma, we have developed for the first time a laboratory model allowing to maintain Neorickettsia sp. through the whole life cycle of a digenean, Plagiorchis elegans. The laboratory life cycle of P. elegans consists of a snail first intermediate host, Lymnaea stagnalis, an aquatic arthropod second intermediate host, Culex pipiens (mosquito larva), and a vertebrate definitive host, Mesocricetus auratus (Syrian hamster). This paper focuses on the development of the laboratory life cycle, as well as outlines its potential uses in studying the transmission biology of Neorickettsia and its evolutionary relationship within its digenean host.

Ultrastructure and development of Nosema podocotyloidis n. sp. (Microsporidia), a hyperparasite of Podocotyloides magnatestis (Trematoda), a parasite of Parapristipoma octolineatum (Teleostei).

Nosema podocotyloidis n. sp. (Microsporidia, Nosematidae) is described from Podocotyloides magnatestis (Trematoda: Opecoelidae), a parasite of the fish Parapristipoma octolineatum (Teleostei) in the Atlantic Ocean. Electron microscopy reveals that all the stages of the cycle (merogony and sporogony) are diplokaryotic and in direct contact with the cytoplasm of host cells. There is no sporophorous vesicle (pansporoblast). The earliest stages observed are meronts, which have a simple plasmic membrane. Their cytoplasm is granular, rich in ribosomes and contains some sacculi of endoplasmic reticulum. They divide by binary fission into diplokaryotic sporonts. The sporonts have a thick electron-dense wall. Their diplokaryon is slightly less electron-dense than the cytoplasm. The cytoplasm of more advanced sporonts has numerous electron-lucent vesicles. Sporonts with two diplokarya divide by binary fission into diplokaryotic sporoblasts. The older sporoblasts are irregular or elongate and the polar filament is in formation. Their cytoplasm is denser, with ribosomes and lamellae of granular endoplasmic reticulum. The sporoblasts evolve into spores. The mature spores are broadly oval and measure 3.6 (3.1-4.0) × 2.58 (1.8-3.3) µm. Their wall is 100-300 nm thick. The polar tube is isofilar with 11-16 coils, 130-155 nm in diameter and arranged in many layers in the centre of the spore. The polaroplast is divided into two regions: an outer electron-dense cup with granular content and lacking lamellae and an internal region, less electron-dense, composed of irregularly arranged sacs. The posterior vacuole, with an amorphous electron-dense content, is present. The new species is compared with other species of Nosema from trematodes.

Large scale screening of digeneans for Neorickettsia endosymbionts using real-time PCR reveals new Neorickettsia genotypes, host associations and geographic records.

Digeneans are endoparasitic flatworms with complex life cycles including one or two intermediate hosts (first of which is always a mollusk) and a vertebrate definitive host. Digeneans may harbor intracellular endosymbiotic bacteria belonging to the genus Neorickettsia (order Rickettsiales, family Anaplasmataceae). Some Neorickettsia are able to invade cells of the digenean's vertebrate host and are known to cause diseases of wildlife and humans. In this study we report the results of screening 771 digenean samples for Neorickettsia collected from various vertebrates in terrestrial, freshwater, brackish, and marine habitats in the United States, China and Australia. Neorickettsia were detected using a newly designed real-time PCR protocol targeting a 152 bp fragment of the heat shock protein coding gene, GroEL, and verified with nested PCR and sequencing of a 1371 bp long region of 16S rRNA. Eight isolates of Neorickettsia have been obtained. Sequence comparison and phylogenetic analysis demonstrated that 7 of these isolates, provisionally named Neorickettsia sp. 1-7 (obtained from allocreadiid Crepidostomum affine, haploporids Saccocoelioides beauforti and Saccocoelioides lizae, faustulid Bacciger sprenti, deropegid Deropegus aspina, a lecithodendriid, and a pleurogenid) represent new genotypes and one (obtained from Metagonimoides oregonensis) was identical to a published sequence of Neorickettsia known as SF agent. All digenean species reported in this study represent new host records. Three of the 6 digenean families (Haploporidae, Pleurogenidae, and Faustulidae) are also reported for the first time as hosts of Neorickettsia. We have detected Neorickettsia in digeneans from China and Australia for the first time based on PCR and sequencing evidence. Our findings suggest that further surveys from broader geographic regions and wider selection of digenean taxa are likely to reveal new Neorickettsia lineages as well as new digenean host associations.

Trematodes enhance the development of the nematode-trapping fungus Arthrobotrys (Duddingtonia) flagrans.

The capability of helminth (nematode and trematode) parasites in stimulating nematode trap and chlamydospore development of the nematophagous fungus Arthrobotrys (formerly Duddingtonia) flagrans was explored. Dead adult specimens of trematodes (the liver fluke Fasciola hepatica and the rumen fluke Calicophoron daubneyi) and nematodes (the ascarid Parascaris equorum and the strongylid Oesophagostomum spp.), as well as their secretory products, were placed onto corn meal agar plates concurrently inoculated with A. flagrans. Trapping organs were observed after 5 d and chlamydospores after 16 d, including in the control plates in the absence of parasitic stimulus. However, our data shows that both nematodes and trematodes increase trap and chlamydospore production compared with controls. We show for the first time that significantly higher numbers of traps and chlamydospores were observed in the cultures coinoculated with adult trematodes. We conclude that both the traps and chlamydospores formation are not only related to nematode-specific stimuli. The addition of secretory products of the trematode C. daubneyi to culture medium has potential for use in the large scale production of chlamydospores.

Transmission rates of the bacterial endosymbiont, Neorickettsia risticii, during the asexual reproduction phase of its digenean host, Plagiorchis elegans, within naturally infected lymnaeid snails.

BACKGROUND: Neorickettsia are obligate intracellular bacterial endosymbionts of digenean parasites present in all lifestages of digeneans. Quantitative information on the transmission of neorickettsial endosymbionts throughout the complex life cycles of digeneans is lacking. This study quantified the transmission of Neorickettsia during the asexual reproductive phase of a digenean parasite, Plagiorchis elegans, developing within naturally parasitized lymnaeid pond snails. METHODS: Lymnaea stagnalis snails were collected from 3 ponds in Nelson County, North Dakota and screened for the presence of digenean cercariae. Cercariae were identified to species by PCR and sequencing of the 28S rRNA gene. Neorickettsia infections were initially detected using nested PCR and sequencing of a partial 16S rRNA gene of pooled cercariae shed from each parasitized snail. Fifty to 100 single cercariae or sporocysts were isolated from each of six parasitized snails and tested for the presence of Neorickettsia using nested PCR to estimate the efficiency at which Neorickettsia were transmitted to cercariae during asexual development of the digenean. RESULTS: A total of 616 L. stagnalis were collected and 240 (39%) shed digenean cercariae. Of these, 18 (8%) were Neorickettsia-positive. Six Neorickettsia infections were selected to determine the transmission efficiency of Neorickettsia from mother to daughter sporocyst and from daughter sporocyst to cercaria. The prevalence of neorickettsiae in cercariae varied from 11 to 91%. The prevalence of neorickettsiae in sporocysts from one snail was 100%. CONCLUSION: Prevalence of Neorickettsia infection in cercariae of Plagiorchis elegans was variable and never reached 100%. Reasons for this are speculative, however, the low prevalence of Neorickettsia observed in some of our samples (11 to 52%) differs from the high prevalence of other, related bacterial endosymbionts, e.g. Wolbachia in Wolbachia-dependent filariid nematodes, where the prevalence among progeny is universally 100%. This suggests that, unlike the Wolbachia-filaria relationship, the Neorickettsia-digenean relationship is not obligatory mutualism. Our study represents the first quantitative estimate of the Neorickettsia transmission through the asexual phase of the digenean life cycle.

New genetic lineages, host associations and circulation pathways of Neorickettsia endosymbionts of digeneans.

Neorickettsia is a genus of intracellular bacteria endosymbiotic in digeneans that may also invade cells of vertebrates and are known to cause diseases of wildlife and humans. Herein, we report results of screening for Neorickettsia of an extensive collection of DNA extracts from adult and larval digeneans obtained from various vertebrates and mollusks in the United States. Seven isolates of Neorickettsia were detected by PCR and sequenced targeting a 527 bp long region of 16S rRNA. Sequence comparison and phylogenetic analysis demonstrated that four isolates matched published sequences of Neorickettsia risticii. Three other isolates, provisionally named "catfish agents 1 and 2" (obtained from Megalogonia ictaluri and Phyllodistomum lacustri, both parasitic in catfishes) and Neorickettsia sp. (obtained from cercariae of Diplostomum sp.), differed from previously known genotypes of Neorickettsia and are likely candidates for new species. All 7 isolates of Neorickettsia were obtained from digenean species and genera that were not previously reported as hosts of these bacteria. Members of four digenean families (Dicrocoeliidae, Heronimidae, Macroderoididae and Gorgoderidae) are reported as hosts of Neorickettsia for the first time. Our study reveals several new pathways of Neorickettsia circulation in nature. We have found for the first time a Neorickettsia from a digenean (dicrocoeliid Conspicuum icteridorum) with an entirely terrestrial life cycle. We found N. risticii in digeneans (Alloglossidium corti and Heronimus mollis) with entirely aquatic life cycles. Previously, this Neorickettsia species was known only from digeneans with aquatic/terrestrial life cycles. Our results suggest that our current knowledge of the diversity, host associations and circulation of neorickettsiae is far from satisfactory.

[Mechanisms for the formation of a combination of the natural foci of trematodiases and tularemia in the floodplain-river ecosystem of the Konda River in different periods of epizootic activity of an infection focus].

The basis of the mechanism responsible for the formation of a combination of the natural foci of trematodiases and tularemia in the infection foci in the interepizootic periods is their association with the morphological structure of the floodplain-river landscape of the Konda River. The landscape's facias and holes, which are common to the foci, are integrated, by predetermining the territorial combination. The formation of a closed trinomial Francisella tularensis parasite system integrating these foci serves as a basis for the mechanism of the formation of a combination of the natural foci of trematodiases and tularemia on transition of the latter from the interepizootic state to epizooty. The absence of host populations linking the foci of methorchiasis (M. xanthosomus), bilharziasis, and tularemia determines their combination both in the interepizootic periods and at the level of the landscape morphological structure. The basis for the mechanism of the formation of a combination of the natural foci of trematodiases and tularemia in the periods of diffuse epizooties in the infection foci is the formation of an open binomial F. tularensis parasite system and the infestation of the co-members of F. tularensis trematode parasite systems. By penetrating into them, the pathogen forces the F. tularensis parasite system to enter the trematode parasite systems, causing their complete (opisthorchiases, methorchiases) or partial (bilharziasis) coincidence. Thus, these foci are combined at the level of parasitocenoses of the parasite systems of infections and infestations, by giving rise to pseudosystem combined foci. The natural foci of B. polonica and F. tularensis are combined at the level of parasitocenosis of the coacting hemipopulation of trematode parthenitas, F. tularensis subpopulations and intermediate host population, by forming an autoecious population-combination bilharziasis-tularemia focus. The formation of territorial-combination, autoecious population-combination, and pseudosystem-combination foci of trematodiases and tularemia in different periods of the epizootic activity of an infection focus is determined by the functional organization of the parasite system of a tularemia focus and the biocenotic (functional) structure of natural foci of infestations.

Neorickettsia helminthoeca and salmon poisoning disease: a review.

Neorickettsia helminthoeca is an obligate intra-cytoplasmic bacterium that causes salmon poisoning disease (SPD), an acute, febrile, fatal disease of dogs. The complex life-cycle of this pathogen involves stages in an intestinal fluke (Nanophyetus salmincola), a river snail (Oxytrema silicula), in fish, and in fish-eating mammals. This complexity has created confusion with respect to the various bacterial and parasitic infections associated with the disease and its significance in dogs in specific geographical locations has likely to have previously been under-estimated. This paper addresses the history, taxonomy, microbiology of N. helminthoeca and summarises the pathogenesis, clinical signs and pathological features associated with infection. Furthermore, the biological cycles, treatment, control, and both public and veterinary health impacts associated with this pathogen and the intestinal fluke N. salmincola are discussed.