Morphological and molecular data confirm the occurrence of Paramphistomum leydeni (Trematoda: Paramphistomidae) in ruminants from Southern Brazil.

Species belonging to the family Paramphistomidae Fischoeder, 1901, commonly known as "rumen flukes", are a group of parasites frequently related to Brazilian livestock production. They inhabit the digestive tract of ruminants and have recognized pathogenicity during the early stages of infection, which can be responsible for economic losses. These trematodes are often associated with Southern Brazil, a region heavily focused on animal farming, which also makes it ideal for the life cycle of paramphistomes. Despite their aforementioned importance, studies regarding their distribution, molecular taxonomy and biology are still scarce in the country. In the present study, rumen flukes collected from cattle (n = 22) and sheep (n = 3) from 9 batches of ruminants from the cities of Jaguarão, Pelotas and Rio Grande, state of Rio Grande do Sul, Brazil, between May and July 2022, were subjected to morphological and molecular study. The microscopic analysis of histological and manual cuts revealed diagnostical traits compatible with Paramphistomum leydeni Näsmark, 1937, including the presence of tegumental papillae, pharynx of the liorchis type and acetabulum of the leydeni type. Molecular data corroborated the morphological identification, with ITS-2 and cox-1 sequences here obtained presenting 100% and 96.8-99.8% similarity, respectively, to P. leydeni samples previously characterized in different countries from Asia, Europe, and South America. Intensity of infection ranged from 5 to 458 and 1 to3 specimens of P. leydeni in sampled cattle and sheep, respectively. The present study contributes to a better understanding of the taxonomy of the flukes involved in cattle and sheep paramphistomosis in Brazil, suggesting that P. leydeni could be the main paramphistome species found in ruminants in the studied region.

Cotylophoron marajoensis n. sp. (Digenea: Paramphistomidae) a parasite of Bubalus bubalis on Marajó Island, Pará, Brazilian Amazon.

The genus Cotylophoron belongs to the Paramphistomidae family and its definitive hosts are ruminants in general. This work describes the presence of a new species of the gender, a parasite in the rumen and reticulum of Bubalus bubalis, on Marajó Island in the Eastern Brazilian Amazon, using of light microscopy, scanning electronic microscopy and molecular biology techniques. One hundred and ten animals were analyzed, of which 4.54% were parasitized by flukes in their adult forms. The helminths were found fixed to the ruminal mucosa and present Liorchis-type pharynx, Cotylophoron-type genital sucker, oblique testicles larger than the ovary, uterus in rings full of eggs and Cotylophoron-type acetabulum. These morphologic characters do not fit into any previously described species. Thus, it is proposed that this is a new species in the genus Cotylophoron. The present work expands the record of parasitism by helminths in Bubalus bubalis, this being the first record of trematoda from the genus Cotylophoron for this host in the Brazilian Amazon.

A phylogenetic study of the cecal amphistome Zygocotyle lunata (Trematoda: Zygocotylidae), with notes on the molecular systematics of Paramphistomoidea.

Zygocotyle lunata inhabits the caecum of birds and mammals from the American continent. This amphistome parasite is easily maintained in the laboratory and serves as a model organism in life-cycle studies, but it has seldom been studied using molecular data. Neither the position of Z. lunata in the superfamily Paramphistomoidea nor the monophyly of the Zygocotylidae has been evaluated with molecular phylogenetic methods. In the present study, adult specimens of Z. lunata obtained experimentally in mice from Brazil were submitted to molecular studies. Partial sequences of nuclear (1261 bp of 28S and 418 bp of 5.8S-ITS-2) and mitochondrial (1410 bp of cytochrome c oxidase 1, cox1) markers were compared with published data. In the most well-resolved phylogeny, based on 28S sequences, Z. lunata clustered in a well-supported clade with Wardius zibethicus, the only other species currently included in the Zygocotylidae, thus confirming the validity of this family. Divergence of 28S sequences between these species was 2.2%, which falls in the range of intergeneric variation (0.9-5.6%) observed in the other two monophyletic groups in the 28S tree, i.e., representatives of Gastrodicidae and Neotropical cladorchiids (Cladorchiidae). Analysis of ITS-2 and two parts of the cox1 gene placed Z. lunata within poorly resolved clades or large polytomies composed of several paramphistomoid families, without clarifying higher-level phylogenetic relationships. The cox1 of a Brazilian isolate of Z. lunata is 99.6% similar to a Canadian isolate, confirming the pan-American distribution of the species. Finally, our phylogenetic reconstructions of Paramphistomoidea revealed a complex scenario in the taxonomic composition of some amphistome families, which highlights a need for further integrative studies that will likely result in rearrangements of traditional morphology-based classifications.

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.

Cotylophoron marajoensis n. sp. (Digenea: Paramphistomidae) a parasite of Bubalus bubalis on Marajó Island, Pará, Brazilian Amazon / Cotylophoron marajoensis n. sp. (Digenea: Paramphistomidae) parasito de Bubalus bubalis na Ilha de Marajó, Pará, Amazônia Brasileira

Abstract The genus Cotylophoron belongs to the Paramphistomidae family and its definitive hosts are ruminants in general. This work describes the presence of a new species of the gender, a parasite in the rumen and reticulum of Bubalus bubalis, on Marajó Island in the Eastern Brazilian Amazon, using of light microscopy, scanning electronic microscopy and molecular biology techniques. One hundred and ten animals were analyzed, of which 4.54% were parasitized by flukes in their adult forms. The helminths were found fixed to the ruminal mucosa and present Liorchis-type pharynx, Cotylophoron-type genital sucker, oblique testicles larger than the ovary, uterus in rings full of eggs and Cotylophoron-type acetabulum. These morphologic characters do not fit into any previously described species. Thus, it is proposed that this is a new species in the genus Cotylophoron. The present work expands the record of parasitism by helminths in Bubalus bubalis, this being the first record of trematoda from the genus Cotylophoron for this host in the Brazilian Amazon.

Resumo O gênero Cotylophoron pertence à família Paramphistomidae e possui como hospedeiros definitivos ruminantes em geral. Este trabalho descreve a presença de uma espécie nova do gênero, parasito do rúmen e retículo de Bubalus bubalis, na Ilha de Marajó, Amazônia oriental brasileira, a partir das técnicas de microscopia de luz, microscopia eletrônica de varredura e biologia molecular. Foram analisados 110 animais, dos quais 4,54% estavam parasitados por trematódeos na sua forma adulta. Os helmintos foram encontrados fixados à mucosa ruminal, apresentando faringe do tipo Liorchis, ventosa genital do tipo Cotylophoron, testículos oblíquos maiores que o ovário, útero em alças repleto de ovos, e acetábulo do tipo Cotylophoron. Estes caracteres morfológicos não se enquadram em nenhuma espécie previamente descrita. Assim, propõe-se uma nova espécie ao gênero Cotylophoron. O presente trabalho amplia o registro do parasitismo por helmintos em Bubalus bubalis, sendo este o primeiro registro de trematódeos do gênero Cotylophoron nesse hospedeiro para a Amazônia brasileira.

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.

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.

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.

A new species of Amphisbaena from the state of Amazonas, Brasil (Reptilia, Amphisbaenia, Amphisbaenidae)

Amphisbaena hugoi, sp. n., from the site of the Balbina hydroelectric dam, in the state of Amazonas (01º53'S, 59º28'W), is sufficiently characterized by having (one specimen known): 4 preanal pores; 225 body annuli; 36 tail annuli, with autotomy constriction on the 13th annulus, 16 dorsal and 18 ventral segments to a midbody annulus; body color patter a brown back and a white belly, clearly demarcated, anteriorly at the level of the lateral sulcus, posteriorly one and then two scales below it, with a thin light line on the lateral sulcus, a white cap on the head, dorsally extending onto the neck; autotomy annulus ventrally brown-colored.