Complex morphological characterization and morphometric-molecular discrimination of two paramphistome species co-infecting cattle, Orthocoelium sp. and Paramphistomum epiclitum.

Co-infection by two paramphistome species, Orthocoelium sp. and Paramphistomum epiclitum, is found in cattle in Thailand. The morphological features of these and other paramphistomes under a light microscope are similar, resulting in misidentification and misdiagnosis. We classified these paramphistomes into three morphological variation types, namely Orthocoelium sp., P. epiclitum MV1 (immature), and P. epiclitum MV2 (matured). Ten morphological characteristics were investigated, and the values were transformed into 25 ratio criteria for statistical investigation. Morphometric analysis can classify the variation of these specimens using differences in the bifurcal level, the vitellaria starting level, the starting level of the anterior testis, and the center level of the posterior testis positions by body length ratios. These ratios can separate the samples into three morphologically different groups, whereas molecular analysis based on the nuclear internal transcribed spacer 2 (ITS2) region and the mitochondrial cytochrome c oxidase subunit I (COI) gene could only distinguish two specific groups. In addition, the Orthocoelium specimen, related to O. dicranocoelium and O. parvipapillatum according to morphological and histological analysis, was monophyletic grouped via ITS2 analysis. Our study provides a scientific basis for the taxonomic classification and clustering of morphologically varying species, improving the identification, detection, and diagnosis of co-infecting paramphistomes.

Morphological and Molecular Identification of Paramphistomum epiclitum from Buffaloes in Pakistan.

INTRODUCTION: Little is known about the genetic and morphological characters of Paramphistomum epiclitum. For the first time in Pakistan, adult flukes were morphologically characterized and the sequence variation in the nuclear ribosomal DNA (rDNA) region, including the first internal transcribed spacers (ITS1) and the 5.8S gene of the Paramphistomum epiclitum were studied. METHODS: Adult amphistomes were examined by light microscopy and sequences of ITS1 and 5.88S rDNA genes were obtained. RESULTS: Twenty adult flukes were measured, 13.17 ± 1.19 mm in length and 5.28 ± 1.34 mm in width. Seventeen adult flukes were sequenced and high sequence variability was observed in 5' end of ITS1 region. The 5.8S and 3' end of ITS1 sequences had 100% identity among the samples. A comparative analysis revealed that different types and numbers of repeats were found within each ITS1 region. The 3' end of ITS1 region from P. epiclitum showed 98% homology with P. cervi from China and formed a subclade with genetic distance of 0.1663. The 5.8S gene showed 100% identity within Paramphistomidae family and formed a sub-clade with P. epiclitum, P. leydeni, P. cervi and Cotylophoron cotylophorum species isolated from China, India and Uruguay. CONCLUSION: This work provides new information on morphological identity and genetics of P. epiclitum from Pakistan.

Morphological and molecular identification with frequency analysis of Calicophoron microbothrioides in central Chile / Identificação morfológica e molecular com análise de frequência de Calicophoron microbothrioides na região central do Chile

Abstract This research aimed to determine the presence of paramphistomids in cattle slaughtered in a slaughterhouse of the Ñuble Region of Chile, to identify flukes and to analyze the frequency of these parasites in the Maule, Ñuble, and Biobío administrative regions of Chile. Between October of 2016 and April of 2017, rumens of 494 cattle were examined for flukes in the forestomachs. Worms were identified morphologically and, in addition, molecular analysis of the internal transcriber spacer region 2 of the fluke's DNA was done and phylogenetic analyses were performed with Bayesian inference in 14 worms. The frequency was analyzed by locality (low- or highlands) and age. The overall frequency was 11.24%. The district with the highest frequency of presentation was Chillán Viejo (30.8%). Districts in the lowlands had similar frequencies to those in the mountain lands (p=0.1). The frequency of flukes was significantly higher in adult animals than in young ones (p<0.01). We obtained a 460 bp-length fragment of DNA that was identical to the sequences previously identified as Paramphistomum cervi and Calicophoron microbothrioides, and performed morphological analyses confirmed that our samples belonged to C. microbothrioides. This is the first published study of C. microbothrioides in Chile.

Resumo Este trabalho teve como objetivo determinar a presença de paramphistomídeos em bovinos abatidos em um matadouro da Região do Ñuble do Chile, para identificar parasitas e analisar a frequência desses parasitos nas regiões administrativas de Maule, Ñuble e Biobío, no Chile. Entre outubro de 2016 e abril de 2017, rúmens de 494 bovinos foram examinados à procura de vermes no pré-estômago. Os vermes foram identificados morfologicamente e, além disso, a análise molecular da região interna do espaçador do transcritor 2 do DNA e análises filogenéticas foram realizadas com inferência bayesiana em 14 vermes. A frequência foi analisada pela altitude da localidade (baixa ou alta) e idade. A frequência geral foi de 11,24%. O distrito com as maiores frequências de parasitismo foi Chillán Viejo (30,8%). Os distritos das terras baixas tinham frequências semelhantes às encontradas nas terras das montanhas (p=0,17). A frequência foi significativamente maior em animais adultos do que em jovens (p<0.01). Obtivemos um fragmento de DNA de 460 pb que era idêntico às sequências anteriores identificadas como Paramphistomum cervi e Calicophoron microbothrioides, e realizamos análises morfológicas que permitiram confirmar que nossas amostras pertenciam a C. microbothrioides. Este é o primeiro estudo publicado sobre C. microbothrioides no Chile.

Morphological and molecular identification with frequency analysis of Calicophoron microbothrioides in central Chile.

This research aimed to determine the presence of paramphistomids in cattle slaughtered in a slaughterhouse of the Ñuble Region of Chile, to identify flukes and to analyze the frequency of these parasites in the Maule, Ñuble, and Biobío administrative regions of Chile. Between October of 2016 and April of 2017, rumens of 494 cattle were examined for flukes in the forestomachs. Worms were identified morphologically and, in addition, molecular analysis of the internal transcriber spacer region 2 of the fluke's DNA was done and phylogenetic analyses were performed with Bayesian inference in 14 worms. The frequency was analyzed by locality (low- or highlands) and age. The overall frequency was 11.24%. The district with the highest frequency of presentation was Chillán Viejo (30.8%). Districts in the lowlands had similar frequencies to those in the mountain lands (p=0.1). The frequency of flukes was significantly higher in adult animals than in young ones (p<0.01). We obtained a 460 bp-length fragment of DNA that was identical to the sequences previously identified as Paramphistomum cervi and Calicophoron microbothrioides, and performed morphological analyses confirmed that our samples belonged to C. microbothrioides. This is the first published study of C. microbothrioides in Chile.

Loads of trematodes: discovering hidden diversity of paramphistomoids in Kenyan ruminants.

Paramphistomoids are ubiquitous and widespread digeneans that infect a diverse range of definitive hosts, being particularly speciose in ruminants. We collected adult worms from cattle, goats and sheep from slaughterhouses, and cercariae from freshwater snails from ten localities in Central and West Kenya. We sequenced cox1 (690 bp) and internal transcribed region 2 (ITS2) (385 bp) genes from a small piece of 79 different adult worms and stained and mounted the remaining worm bodies for comparisons with available descriptions. We also sequenced cox1 and ITS2 from 41 cercariae/rediae samples collected from four different genera of planorbid snails. Combining morphological observations, host use information, genetic distance values and phylogenetic methods, we delineated 16 distinct clades of paramphistomoids. For four of the 16 clades, sequences from adult worms and cercariae/rediae matched, providing an independent assessment for their life cycles. Much work is yet to be done to resolve fully the relationships among paramphistomoids, but some correspondence between sequence- and anatomically based classifications were noted. Paramphistomoids of domestic ruminants provide one of the most abundant sources of parasitic flatworm biomass, and because of the predilection of several species use Bulinus and Biomphalaria snail hosts, have interesting linkages with the biology of animal and human schistosomes to in Africa.

Molecular identification of the rumen flukes Paramphistomum leydeni and Paramphistomum cervi in a concurrent infection of the red deer Cervus elaphus.

Paramphistomosis, caused by paramphistomid flukes, is a gastrointestinal parasitic disease of domestic and wild ruminants. Originally thought to be limited to the tropics and subtropics, the disease has recently been reported in temperate regions. Here we describe the concurrent infection of a red deer doe (Cervus elaphus) with Paramphistomum leydeni and Paramphistomum cervi. This is the first report of P. leydeni in Croatia. Flukes were identified on the basis of morphological keys (tegumental papillae) and sequencing of the internal transcribed spacer region 2 in ribosomal DNA. Our results confirm that the absence of tegumental papillae allows P. cervi to be differentiated morphologically from other paramphistomid species in Europe based on incident light stereomicroscopy. Nevertheless the limitations of morphological identification and taxonomic issues suggest that previous findings on paramphistomid infection should be interpreted carefully. The possible worldwide distribution of these pathogens means that paramphistomosis may be more common and its economic impact greater than previously thought.

First Record of Paramphistomes Fischoederius cobboldi and Paramphistomum epiclitum Detected in Bovine Rumen from a Local Market of Savannakhet Province, Lao PDR.

In the present study, we report on the occurrence of paramphistomes, Fischoederius cobboldi and Paramphistomum epiclitum, in Lao PDR with the basis of molecular data. Parasite materials were collected from bovines bred in Ban Lahanam area, Savannakhet Province, Lao PDR at Lahanam public market. Morphological observations indicated 2 different species of paramphistomes. The mitochondrial gene cox1 of the specimens was successfully amplified by PCR and DNA sequencing was carried out for diagnosis of 11 specimens. Pairwise alignment of cox1 sequences were performed and confirmed F. cobboldi and P. epiclitum infecting bovines in Laos. Although there were many limiting points, as the small number of worm samples, and the restricted access of the animal host materials, we confirmed for the first time that 2 species of paramphistomes, F. cobboldi and P. epiclitum, are distributed in Lao PDR. More studies are needed to confirm the paramphistome species present in Savannakhet and its hosts to clear the natural history of these parasites of ruminants in the region and measure the impact of this parasite infection in the life and health of the local people.

Descripción morfológica de Cotylophoroncotylophorum (Digenea: Paramphistomidae) hallado enbovinos de Rionegro, Antioquia, Colombia / Descrição morfológica do Cotylophoron cotylophorum (Digenea: Paramphistomidae)em bovinos de Rionegro, Antioquia, Colômbia / Morphological description of Cotylophoron cotylophorum (Digenea: Paramphistomidae) in Cattle from Rionegro, Antioquia, Colombia

En Colombia se ha registrado la presencia de Paramphistomidae (Trematoda: Digenea) parasitandoel rumen de bovinos de la costa Caribe, los Llanos Orientales y el suroccidente. En estas publicacionesse menciona la presencia y prevalencia de los paramfistómidos, pero todas carecen de la descripciónmorfológica de los digeneos. El hallazgo de paramfistómidos adultos en el rumen de ejemplaresBos taurus, procedentes del sector Llanogrande, en el municipio de Rionegro, Antioquia, motivó lapresente investigación, con el propósito de describir los diferentes estadíos del parásito y establecerel estatus taxonómico específico. Del rumen de una hembra, Bos taurus, sacrificada en la CentralGanadera del municipio de Medellín, se extrajeron vivos 52 digeneos adultos. En el laboratoriose aislaron huevos que se incubaron hasta la obtención de miracidios. Con ellos se infectaron 50moluscos Lymnaea truncatula. La morfología de los adultos y estadíos larvarios del paramfistómidopermitió determinarlo como Cotylophoron cotylophorum. En L. truncatula se obtuvo una infeccióndel 5.1 %. Con este registro se amplia la distribución geográfica y altitudinal del parásito a la regiónaltoandina, noroccidental de Colombia.

The presence of Paramphistomidae parasites (Trematoda: Digenea) inside the rumen (first stomach) hasbeen recorded in Colombia in cattle from the Caribbean coast, the Eastern Plains region in the east, and thesouthwest of the country. However, studies on the presence and prevalence of the parasite do not include anymorphological description. This study includes research findings from adult Paramphistomidae parasitesin the rumen of Bos taurus specimens from Llanogrande in the municipality of Rionegro, Antioquia. Theaim of this research was to describe all the developmental stages of the parasite and to establish its specifictaxonomical status. 52 live adult parasites were found in the rumen of a female Bos taurus sacrificed inthe slaughter house Central Ganadera of Medellin. Eggs from adults were obtained in the laboratory, andhatching was stimulated to obtain miracidia. 50 Lymnaea truncatula snails were exposed to the miracidia,resulting in an infection rate of 5.1 %. The parasite was identified as Cotylophoron cotylophorum byusing adult morphology and larval stages. This finding expands the parasite’s geographical and altitudinaldistribution to the andean highlands in northwestern Colo.

Na Colômbia tem-se encontrado a presencia de Paramphistomidae (Trematoda: Digenea) parasitandono rúmen de bovinos da costa Caribe, os Llanos Orientales e no sul oeste. Nestas publicações tem-semencionado a presencia e prevalência dos paramfistómidos, mas todas carecem da descrição morfológicados digeneos. Do rúmen de uma fêmea Bos taurus proveniente do município de Rionegro e sacrificadana Central Ganadera do município de Medellín, se extraíram vivos 52 digeneos adultos. No laboratórioforam isolados os ovo e forma obtidos os miracidios. Com eles foram infetados 50 moluscos Lymnaeatruncatula. A morfologia dos adultos e estádios larvários do paramfistómido permitiu determinar-lho comoCotylophoron cotylophorum. Em L. truncatula foi obtido uma infecção de 5.1 %. Com este fato, se ampliaa distribuição geográfica e altitudinal do parasita na região alta dos Andes colombianos.

[Gastrodiscoidosis is a dangerous zoonosis].

The paper gives data on the helminthiasis--gastrodiscoidosis, a zoonotic disease caused by the trematode Gastrodiscoides hominis (Lewis et McConnall, 1876) parasitizing in the animal and human intestine in the endemic foci of both Russia and foreign countries. It also presents information on the morphology of the helminth, the biological cycle of development of the parasite and its habitat and spread, as well as its induced abnormalities and on the method of diagnosing of the disease.

Natural infection by Paramphistomoidea Stiles and Goldberger, 1910 trematodes in wild Marsh Deer (Blastocerus dichotomus Illiger, 1815) from Sérgio Mottas's hydroelectric power station flooding area.

Studies on helminthfauna of marsh deer Blastocerus dichotomus Illiger, 1815 are rare, although helminthic diseases are an important cause of mortality in these animals. Fifteen male and female adult marsh deer from Sergio Motta's hydroelectric power station flooding area at Paraná River which died during the capture and quarantine procedures, between 1998 and 1999, were necropsied. Three trematodes species, Paramphistomum cervi, Balanorchis anastrofus and Zygocotyle lunatum, all belonging to superfamily Paramphistomoidea, were identified. The obtained trematodes were identified, counted and their respectives descriptors of infection were determined. All necropsied animals were infected by helminths. Paramphistomum cervi was the most prevalent species, while Zygocotyle lunatum was found in only one animal.

In vitro studies on the effects of some anthelmintics on Cotylophoron cotylophorum (Digenea, Paramphistomidae): a structural analysis.

The effects of the anthelmintics praziquantel (PZQ), levamisole (LEV), mebendazole (MBZ), fenbendazole (FBZ) and albendazole (ABZ), on the morphology and the histology of a digenetic trematode, Cotylophoron cotylophorum, were studied. Scanning electron micrographs of the drug-treated worms revealed that PZQ was the most effective drug inducing surface damages to a great extent. The parasite exposed to PZQ for 6 h, showed smaller blebs on the oral sucker region as well as on the sensory papillae. These blebs enlarged in size after 24 h and ruptured after 30 h of exposure. The worms treated with LEV showed a few smaller blebs on the ventrolateral margin. In MBZ- and FBZ-treated worms the blebs appeared between the oral and genital sucker after 6 h of incubation. The changes were not apparent in the ABZ-treated worms.

Paramphistomum daubneyi: the development of redial generations in the snail Lymnaea truncatula.

Rediae of Paramphistomum daubneyi were counted and measured in Lymnaea truncatula to elucidate the variability in the numbers of free rediae and cercariae occurring between naturally infected snails and experimental single-miracidium infections. Experiments were performed using one miracidium per snail and snail raising was carried out at 20 degrees C. Two redial generations succeeded each other in the snail until day 49. A mean of 8-10 rediae differentiated in the sporocyst at days 7 and 14; the remaining germ balls and redial embryos decreased in number after day 21. First-generation rediae became free in the snail's body starting at day 14 and their number increased to a mean of 7.5 at day 49, with a maximum of 10 rediae being detected in 1 snail. They produced second-generation rediae, which exited from the body starting at day 28, and then cercariae, which exited from day 42 onward. The count of second-generation rediae was 6-6.2/snail at day 49, with a maximum of 12 being detected in 1 snail; they produced only cercariae.

Catadiscus pomaceae sp. n. (Trematoda, Paramphistomatidae) from Pomacea canaliculata (Lamarck, 1801) (Prosobranchia, Ampullariidae)

Catadiscus pomaceae sp. n. from the intestine of the prosobranch mollusc Pomacea canaliculata (Lamarck, 1801), is described. The host snail was collected from a lenitic biotope belonging to the Riachuelo basin (Corrientes province, Argentina) during 1985-1986. So far the species of the genus Catadiscus Cohn, 1904 have been recorded in amphibians and reptiles. This is the first instance of a species of that genus parasitizing a mollusc

Cotylophoron Travassosi sp. n. (Trematoda--Paramphistomidae) from cattle.

A new species of the genus Cotylophoron (Trematoda--Paramphistomidae) --Cotylophoron travassosi sp. n.--is described. The measurements of the worm and its structures are compared with the valid known species.

Catadiscus pomaceae sp. n. (Trematoda, Paramphistomatidae) from Pomacea canaliculata (Lamarck, 1801) (Prosobranchia, Ampullariidae).

Catadiscus pomaceae sp. n. from the intestine of the prosobranch mollusc Pomacea canaliculata (Lamarck, 1801), is described. The host snail was collected from a lenitic biotope belonging to the Riachuelo basin (Corrientes province, Argentina) during 1985-1986. So far the species of the genus Catadiscus Cohn, 1904 have been recorded in amphibians and reptiles. This is the first instance of a species of that genus parasitizing a mollusc.

Drepanotrema kermatoides (Orbigny, 1835) (Mollusca, Planorbidae), hospedeiro de um paranfistomídeo (Trematoda), no Rio Grande do Sul, Brasil / Drepanotrema kermatoides (Orbigny, 1835) (Mollusca, Planorbidae), host of a paramphistomid (Trematoda), in Rio Grande do Sul, Brazil

Säo descritas e ilustrradas rédia, cercária imatura e cercária madura e metacercária de um paranfistomídeo, provavelmente do gênero Paramphistomum (Fischoeder, 1901). Estas formas evolutivas foram obridas de um exemplar de Drepanotrema Kermatoides (Orbigny, 19835) coletado, em maio de 1983, no arroio Chico Lumä (29-56'03"S e 50-35'59"W), município de Santo Antônio da Patrulha, Rio Grande do Sul, Brasil

[The first finding of Paramphistomum daubneyi (Dinnik, 1962) in beef cattle in Algeria]. / Prvý nález Paramphistomum daubneyi (Dinnik, 1962) u hovädzieho dobytka na území Alzírska.

The first finding of the causative agent of paramphistomatosis of cattle in the coastal region of Algeria is described. As found on the basis of histological diagnosis, this causative agent is the species Paramphistomum daubneyi (Dinnik, 1962). Lymnaea truncatula was found to be present as a potential intermediate host at the localities where the cattle harbouring these trematodes were kept. It is suggested by the high intensity of invasion by the trematodes (up to 2204 specimens in one host) that the animals suffered from severe helminthosis in some cases. The finding of the causative agent of bovine paramphistomatosis in Algeria draws attention to the need of further investigation of trematodosis in the cattle kept in that country.

[Chaetotaxy of the cercaria of Paramphistomum sp., a parasite of cattle in Vendée]. / Chétotaxie de la cercaire de Paramphistomum sp. parasite du bétail en Vendée.

The cercaria was shed by an Anisus rotundatus experimentally infected with a parasite of Bos taurus in Vendée (France). This Trematode was identified as Paramphistomum cervi. The snail natural host is unknown. The chaetotaxy of the cercaria differs from that of other European species of Paramphistomum and particularly from P. leydeni and P. daubneyi both species which occur in Vendée. The morphology of the adult precludes any relationship with P. ichikawai also present in Vendée. Therefore it very likely represents a species which was synonymised with P. cervi or a new species.