Genetic diversity of Hepatozoon spp. in rodents from Chile / Diversidade genética de Hepatozoon spp. em roedores do Chile

Abstract This study aimed to investigate the genetic diversity of Hepatozoon spp. in rodents from Valdivia, Chile. A total of 74 rodents (synanthropic n=38; wild n=36) were trapped in Valdivia. We performed conventional PCR assays for Apicomplexa organisms targeting two overlapping 18S rDNA gene fragments (600 bp and 900 bp) followed by sequencing of selected amplicons. Hepatozoon spp. occurrence was 82.43% (61/74). Twelve sequences obtained from the 600 bp and ten from the 900 bp 18S rDNA fragments were identified as Hepatozoon sp. Six sequences obtained from 18S rDNA-based overlapping PCR protocols were used for concatenated (1,400 bp) phylogenetic, haplotype and distance analyses. Hepatozoon spp. 18S rDNA concatenated sequences from the present study were detected in Oligoryzomys longicaudatus, Rattus norvegicus, Mus musculus, and Abrothrix longipilis grouped with Hepatozoon species earlier described in rodents and reptiles from Chile and Brazil. Nucleotide polymorphism of the six 18S rDNA sequences (1,400 bp) from this study, and other Chilean sequences from rodents and rodent's ticks, showed high diversity with a total of nine Chilean haplotypes. Three haplotypes from Valdivia were identified for the first time in this study, suggesting the circulation of novel haplotypes in rodents from southern Chile.

Resumo Este estudo teve como objetivo investigar a diversidade genética de Hepatozoon spp. em roedores de Valdivia, Chile. Um total de 74 roedores (sinantrópicos n=38; selvagens n=36) foram capturados. PCR convencional foi realizada para organismos Apicomplexa, visando dois fragmentos sobrepostos do gene 18S rDNA (600 bp e 900 bp), seguida pelo sequenciamento de amplicons selecionados. A ocorrência de Hepatozoon spp. foi de 82,43% (61/74). Doze sequências obtidas dos fragmentos de 18S rDNA de 600 pb e dez dos fragmentos de 18S rDNA de 900 pb foram identificadas como Hepatozoon sp. Seis sequências obtidas, a partir de protocolos de PCR sobrepostos, foram usadas para análises filogenéticas (1.400 bp), de haplótipos e de distância. Sequências concatenadas 18S rDNA do presente estudo foram detectadas em Oligoryzomys longicaudatus, Rattus norvegicus, Mus musculus e Abrothrix longipilis e agrupadas com Hepatozoon descrito em roedores e répteis do Chile e do Brasil. A análise de polimorfismos das seis sequências deste estudo, junto com outras sequências chilenas de roedores e carrapatos de roedores, mostrou alta diversidade com um total de nove haplótipos no Chile. Três haplótipos detectados em Valdivia foram identificados pela primeira vez neste estudo, sugerindo que novos haplótipos circulam em roedores do sul do Chile.

Sarcocystidae in wild birds of southeastern Brazil / Sarcocystidae em aves silvestres do sudeste do Brasil

Abstract This study aimed to identify members of the Sarcocystidae family in naturally infected wild birds at a rescue center in the state of Minas Gerais, southeastern Brazil. The heart and brain of 44 wild birds were evaluated by bioassay in mice to detect T. gondii, and extracted DNA was used for nested PCR of the 18S ribosomal DNA gene to detect members of the Sarcocystidae family. The positive samples were sequenced, assembled, edited and compared with sequences deposited in GenBank. Toxoplasma gondii was isolated from six (13.6%) out of 44 birds. Toxoplasma gondii DNA was identified in 10/44 (22.7%) of the birds. The amplified sequences exhibited 100% similarity with the DNA of the ME49 strain of T. gondii. Sarcocystis DNA (99% similarity) was identified in 5/44 (11.4%) of the birds. T. gondii and Sarcocystis spp. are common in wild birds in Minas Gerais, Brazil.

Resumo O objetivo deste estudo foi identificar membros da família Sarcocystidae em aves silvestres de vida livre naturalmente infectadas e resgatadas no estado de Minas Gerais, Brasil. Coração e cérebro de 44 aves silvestres foram avaliados por bioensaio em camundongos para detecção de T. gondii e extração de DNA para Nested-PCR do gene 18S do DNA ribossomal de membros da família Sarcocystidae. As amostras positivas foram sequenciadas, analisadas, editadas e comparadas com sequências depositadas no GenBank. Toxoplasma gondii foi isolado de seis (13,6%) das 44 aves. DNA de T. gondii foi identificado em 10/44 (22,7%) das 44 aves. As sequências amplificadas exibiram 100% de similaridade com o DNA da cepa ME49 de T. gondii. DNA de Sarcocystis (99% de similaridade) foi identificado em 5/44 (11,4%) das 44 aves. T. gondii e Sarcocystis spp. são encontrados, comumente, em aves silvestres no estado de Minas Gerais, Brasil.

Molecular Detection of Tick-borne Pathogens in Ticks Collected from Hainan Island, China / 生物医学与环境科学（英文）

Pathogens like bacteria and protozoa, which affect human and animal health worldwide, can be transmitted by vectors like ticks. To investigate the epidemiology and genetic diversity of bacteria and protozoans carried by ticks in Chengmai county of Hainan province, China, 285 adult hard ticks belonging to two species [

Molecular evaluation of piroplasms and hematological changes in canine blood stored in a clinical laboratory in Niterói, Rio de Janeiro / Avaliação molecular de piroplasmídeos e alterações hematológicas em amostras de sangue de cães armazenadas em laboratório de análises clínicas de Niterói, Rio de Janeiro

Abstract Piroplasm species were analyzed by molecular tools in total 31 blood samples from positive dogs, previously checked by stained slides, stored until DNA extraction between 2016 to 2018 in the laboratory Clinical Analyzes in Niterói, Rio de Janeiro. The piroplasms were identified by PCR, targeting the 18S rRNA gene and sequencing. From the total number of samples only 24 (77.4%) were positive and show adequate nucleotide sequences for interpretation with identity between 93%-100% with Babesia vogeli in compared to the sequences isolated of infected dogs from other states in Brazil deposited on GenBank. Most of dogs infected with B. vogeli had anemia (62.5%) and thrombocytopenia (95.8%). The findings of this study are compatible with previous reports in the literature and highlight B. vogeli as the most incriminated species in canine piroplasmosis in Brazil, and thrombocytopenia the hematological alteration most frequently identified in this infection. It is important to note that this is the first study involving the molecular characterization of piroplasms in the metropolitan region of Rio de Janeiro, based on PCR followed by sequencing.

Resumo Espécies de piroplasmídeos foram analisadas por meio de métodos moleculares, em 31 amostras de sangue de cães, previamente verificadas em lâminas coradas, estocadas até a extração de DNA entre 2016 a 2018 em laboratório de Análises Clínicas, em Niterói, Rio de Janeiro. Os piroplasmídeos foram identificados pela PCR, utilizando-se como alvo o gene 18S RNAr e, posteriormente, o sequenciamento. Do total de amostras analisadas, somente 24 (77,4%) foram positivas e apresentaram sequências nucleotídicas adequadas para interpretação com identidade variando entre 93% a 100% com B. vogeli, em comparação com as sequências isoladas de cães infectados de outros estados do Brasil, depositadas no GenBank. A maioria das amostras de sangue dos cães detectados com B. vogeli apresentaram, no hemograma, anemia (62,5%) e trombocitopenia (95,8%). Os resultados detectados neste estudo estão compatíveis com o evidenciado na literatura, pois B. vogeli tem sido a espécie mais relatada nas infecções caninas no Brasil, sendo a trombocitopenia a alteração hematológica mais evidenciada nas amostras analisadas. É importante ressaltar que este é o primeiro estudo envolvendo análise molecular e caracterização de piroplasmídeos, em amostras de sangue de cães da região metropolitana do Rio de Janeiro, utilizando-se a PCR associada ao sequenciamento.

[RETRACTED ARTICLE] Neoechinorhynchus macrospinosus (Acanthocephala: Neoechinorhynchidae) in Rabbit fish Siganus rivulatus (Siganidae): morphology and phylogeny / [ARTIGO RETRATADO] Neoechinorhynchus macrospinosus (Acanthocephala: Neoechinorhynchidae) em peixes-coelho Siganus rivulatus (Siganidae): morfologia e filogenia

Abstract Siganids are the most important marine fish distributed along the African coast. Therefore, the current study aimed to investigate parasite fauna infects one of the most important mariculture fish species in the Red Sea, the Rabbit fish Siganus rivulatus. One acanthocephalan species has been isolated from the posterior region of fish intestine, belonging to the Neoechinorhynchidae family, and named as Neoechinorhynchus macrospinosus Amin & Nahhas, 1994 based on its morphological and morphometric features. In order to determine the accurate taxonomic position of this acanthocephalan species, molecular phylogenetic analysis was carried out based on the partial sequences of 18S rDNA gene region. The obtained data revealed that this species was associated with a close identity ˃71% for other species belonging to the Neoechinorhynchidae family. In addition, the recovered species deeply embedded in the Neoechinorhynchus genus, closely related to the previously described Neoechinorhynchus sp., N. mexicoensis, and N. golvani with identity percent of 95.14, 93.59, 93.59%, respectively. Therefore, the present study provide a better understanding about the taxonomic status of N. macrospinosus based on 18S rDNA that can be useful for achieving a proper assessment of biodiversity.

Resumo Os siganídeos são os peixes marinhos mais importantes distribuídos ao longo da costa africana. Portanto, o presente estudo teve como objetivo investigar a fauna de parasitas infectando uma das espécies mais importantes de peixes para maricultura no Mar Vermelho, o peixe-coelho Siganus rivulatus. Uma espécie de acantocéfalo foi isolada da região posterior do intestino de peixes pertencentes à família Neoechinorhynchidae, e denominadas Neoechinorhynchus macrospinosus Amin & Nahhas, 1994, com base em suas características morfológicas e morfométricas. A fim de determinar a posição taxonômica precisa dessa espécie de acantocéfalo, a análise filogenética molecular foi realizada com base nas sequências parciais da região do gene 18S rDNA e revelou que essa espécie estava associada a uma identidade próxima de até 71% para outras espécies pertencentes a família Neoechinorhynchidae e profundamente enraizada no gênero Neoechinorhynchus, intimamente relacionada a Neoechinorhynchus sp., N. mexicoensis, and N. golvani descrito anteriormente com percentual de identidade de 95,14, 93,59, 93,59%, respectivamente. Portanto, o presente estudo fornece uma melhor compreensão sobre o status taxonômico de N. macrospinosus com base no 18S rDNA que pode ser útil para obter uma avaliação adequada da biodiversidade.

First record of Pseudoterranova decipiens (Nematoda, Anisakidae) infecting the Red spot emperor Lethrinus lentjan in the Red Sea / Primeiro registro de Pseudoterranova decipiens (Nematoda, Anisakidae) infectando o imperador da mancha vermelha Lethrinus lentjan no Mar Vermelho

Abstract The current parasitological study was carried out to investigate helminth parasites infecting the Red spot emperor Lethrinus lentjan inhabiting Hurghada City at the Gulf of Suez, Red Sea, Egypt. Third-stage larvae of nematode parasite was isolated from the intestine as well as body cavity of the examined fish. Light and scanning electron microscopy revealed that this parasite belonged to Anisakidae family within the genus Pseudoterranova. The present species is named Pseudoterranova decipiens based on the presence of triangular mouth aperture with prominent boring teeth and soft swellings of the cuticle, long muscular esophagus, ventrally excretory pore, and narrow transverse slit of anal opening followed by a short mucron. The morphological characteristics of this species were confirmed by molecular analysis of 18S rDNA gene region of the present parasite. It demonstrated a close identity ≥89% with taxa under family Anisakidae, 85% with Raphidascarididae, and 79-84% with Toxocaridae. A preliminary genetic comparison between gene sequence of the present parasite and other oxyurid species placeed it as a putative sister taxon to other Pseudoterranova decipiens described previously. This study demonstrated that the 18S rDNA gene region of Pseudoterranova decipiens yielded a unique sequence that confirmed its taxonomic position in Anisakidae.

Resumo O presente estudo parasitológico foi realizado para investigar os helmintos parasitos que infectam o peixe imperador Lethrinus lentjan, que habita a cidade de Hurghada no Golfo de Suez, Mar Vermelho, no Egito. Larvas de terceiro estágio de parasitos nematoides foram isoladas do intestino e da cavidade do corpo do peixe examinado. Microscopia eletrônica de luz e de varredura revelou que este parasita pertence à família Anisakidae dentro do gênero Pseudoterranova. A espécie atual é denominada Pseudoterranova decipiens baseada na presença de abertura triangular da boca com dentes proeminentes chatos e inchaços moles da cutícula, esôfago muscular longo, poro ventralmente excretor e fenda transversal estreita da abertura anal seguida por um mucron curto. As características morfológicas desta espécie foram confirmadas pela análise molecular da região do gene 18S rDNA do presente parasito. Demonstrou uma identidade próxima ≥89% com taxa sob família Anisakidae, 85% com Raphidascarididae, e 79-84% com Toxocaridae. Uma comparação genética preliminar entre a sequência genética do presente parasito e outras espécies de oxiurídeos coloca-o como um taxon irmão putativo para outros Pseudoterranova descritos anteriormente. Este estudo demonstra que a região do gene 18S rDNA de Pseudoterranova decipiens produz uma sequência única que confirma sua posição taxonômica em Anisakidae.

Molecular survey of tick-borne pathogens in small mammals from Brazilian Amazonia / Levantamento molecular de patógenos transmitidos por carrapatos em pequenos mamíferos da Amazônia brasileira

Abstract Small non-volant mammals (marsupials and small rodents) were captured at three different timepoints from 23 forest fragments across three municipalities (Alta Floresta, Sinop and Cláudia) covering the Amazonian biome of the Mato Grosso State in Midwestern Brazil. The animal tissues (liver and spleen) and blood were screened using molecular tools for the detection of Babesia, Coxiella, Cytauxzoon, Hepatozoon, Theileria, and Anaplasmataceae agents. A total of 230 specimens (78 rodents and 152 marsupials) were trapped. Hepatozoon and Piroplasmorida agents were detected in the common opossums (Didelphis marsupialis). In turn, all samples (blood, liver, or spleen) collected from the small mammals were negative for the genus Coxiella and the family Anaplasmataceae, as detected by polymerase chain reaction (PCR). Phylogenetic analyses inferred from partial sequences of the 18S rRNA gene highlighted the occurrence of new Hepatozoon and Piroplasmorida haplotypes. Future studies determining the role of common opossum (D. marsupialis) in the epidemiological cycles of Hepatozoon and Babesia under natural conditions in the Amazonian biome are necessary.

Resumo Pequenos mamíferos não voadores (marsupiais e pequenos roedores) foram capturados em três diferentes períodos, ao longo de 23 fragmentos florestais de três municípios (Alta Floresta, Sinop e Cláudia), localizados no bioma amazônico do Estado de Mato Grosso, no centro-oeste do Brasil. Os tecidos dos animais (fígado e baço) e sangue foram selecionados e submetidos a ensaios moleculares para a detecção do DNA de Babesia, Coxiella, Cytauxzoon, Hepatozoon, Theileria e agentes Anaplasmataceae. Um total de 230 espécimes (78 roedores e 152 marsupiais) foram capturados. Hepatozoon e agentes Piroplasmorida foram detectados em gambás (Didelphis marsupialis). Ao contrário, todas as amostras (sangue, fígado ou baço) coletadas dos pequenos mamíferos foram negativas para o gênero Coxiella e a família Anaplasmataceae, conforme detectado pela reação em cadeia da polimerase (PCR). Análises filogenéticas inferidas pelas sequências parciais do gene 18S rRNA evidenciaram a ocorrência de novos haplótipos de Hepatozoon e Piroplasmorida. Futuros estudos determinando a importância do gambá-comun (D. marsupialis) nos ciclos epidemiológicos de Hepatozoon e Babesia em condições naturais, no bioma amazônico, são necessários.

Molecular detection of Rickettsia genus in chigger mites (Trombidiformes: Trombiculidae) collected on small mammals in southeastern brazilian / Detecção molecular do gênero Rickettsia em ácaros trombiculídeos (Trombidiformes: Trombiculidae) coletados em pequenos mamíferos do sudeste brasileiro

Abstract Chiggers are ectoparasites of vertebrates and may cause trombiculiasis or transmit pathogens to their hosts. Specimens collected from rodents and marsupials were morphologically identified as Herpetacarus hertigi, Eutrombicula tinami, Kymocta sp., Quadraseta brasiliensis, Quadraseta falconensis, Quadraseta flochi, Quadraseta mackenziei, Quadraseta pazca, Quadraseta trapezoides, Quadraseta sp., Serratacarus sp., and Trombewingia bakeri. These mites were submitted individually to molecular analyses for the detection of bacteria of the genus Coxiella, Hepatozoon and Rickettsia. Samples were positive to Rickettsia only. Obtained sequences for the gltA (350 pb) and ompA (488 pb) genes were identical to "Candidatus Rickettsia colombianensi", a species previously detected in ticks. In addition, molecular identification of mites based on 18S rDNA sequences are provided for H. hertigi, Kymocta sp., Q. brasiliensis, Q. pazca, Q. trapezoides, Quadraseta sp., and T. bakeri for the first time. This is the first report of the detection of a Rickettsia sp. in chigger mites collected on rodents in Brazil.

Resumo Os trombiculídeos são ectoparasitas de vertebrados e podem causar trombiculíase ou transmitir patógenos ao hospedeiro. Exemplares coletados em roedores e marsupiais foram identificados morfologicamente como Herpetacarus hertigi, Eutrombicula tinami, Kymocta sp., Quadraseta brasiliensis, Quadraseta falconensis, Quadraseta flochi, Quadraseta mackenziei, Quadraseta pazca, Quadraseta trapezoides, Quadraseta sp., Serratacarus sp. e Trombewingia bakeri. Estes ácaros foram submetidos individualmente à análise molecular para detecção de bactérias dos gêneros Coxiella, Hepatozoon e Rickettsia. Amostras foram positivas somente para Rickettsia. Sequências obtidas para os genes gltA (350 pb) e ompA (488 pb) foram idênticas à "Candidatus Rickettsia colombianensi", uma espécie anteriormente detectada em carrapatos. Além disso, foram fornecidas sequências de DNA 18S para identificação molecular de H. hertigi, Kymocta sp., Q. brasiliensis, Q. pazca, Q. trapezoides, Quadraseta sp. e T. bakeri. Este é o primeiro registro da detecção de Rickettsia em ácaros trombiculídeos coletados em roedores do Brasil.

Hepatozoon sp. gamonts as an accidental finding in synovial liquid from an injured maned wolf (Chrysocyon brachyurus) in southeastern Brazil / Gamontes de Hepatozoon sp. como achado acidental em líquido sinovial de lobo guará (Chrysocyon brachyurus) no sudeste do Brasil

Abstract A free-living, adult male maned wolf (Chrysocyon brachyurus) was referred to the Governador "Laudo Natel" - FCAV/Unesp veterinary hospital after being found with skin lesions and a fracture on the right pelvic limb, which had to be amputated due to compromised integrity. Around 20 days later, bilateral accentuated swollen on humerus-radius-ulna articulation was observed. The synovial liquid was drained and sent to the laboratory for synovial cytology with Rosenfeld staining that revealed predominantly degenerated neutrophils with karyolytic chromatin associated with intracellular inclusions suggestive of Hepatozoon sp. gametocytes. Blood and synovial liquid samples were submitted to molecular analysis, aiming to amplify the Hepatozoon spp. 18S rRNA gene fragment. Despite the positioning of the found Hepatozoon sequence together with Hepatozoon canis previously detected in domestic carnivores, the BLAST analysis showed only 98% identity with H. canis. To the best of the authors' knowledge, this is the first time a Hepatozoon was detected in the synovial liquid by clinical pathology and molecular analyses.

Resumo Um lobo guará (Chrysocyon brachyurus) adulto, macho, de vida livre foi encaminhado para atendimento no hospital veterinário Governador "Laudo Natel" - FCAV/Unesp após ser encontrado com lesões de pele e fratura em membro pélvico direito, sendo amputado devido a comprometimento da integridade do membro. Aproximadamente 20 dias após a chegada ao hospital, foi notado acentuado aumento de volume bilateral em região de articulação úmero-rádio-ulnar. O líquido sinovial foi drenado e enviado para análise citológica com coloração de Rosenfeld, revelando a presença de neutrófilos degenerados com cromatina cariolítica associados a inclusões intracelulares sugestivas de gametócitos de Hepatozoon sp. Amostras de sangue e líquido sinovial foram submetidas a análises moleculares visando amplificar um fragmento do gene 18S rRNA de Hepatozoon spp. Apesar da sequência de Hepatozoon detectada se posicionar filogeneticamente no mesmo clado que H. canis previamente detectado em carnívoros domésticos, o resultado da análise do BLAST mostrou somente 98% de identidade com H. canis. De acordo com o conhecimento dos autores, esta é a primeira vez que Hepatozoon foi detectado no líquido sinovial por meio de patologia clínica e análises moleculares.

First study of Cryptosporidium spp. occurrence in eared doves (Zenaida auriculata) / Primeiro estudo de ocorrência de Cryptosporidium spp. em pomba-de-bando (Zenaida auriculata)

Abstract Cryptosporidium is a protozoan parasite with a wide range of hosts, including humans. However, only a few Cryptosporidium species have been described in birds (C. meleagridis, C. baileyi, C. galli and C. avium). The aim of this study was to investigate the occurrence of Cryptosporidium spp. in feces of eared doves (Zenaida auriculata), followed by molecular characterization of the parasite. A total of 196 animals of both sexes were trap-captured; the animals were culled and the intestinal contents were collected for DNA extraction. After extraction, a nested-PCR (nPCR), which amplifies a fragment of the 18S rRNA gene of Cryptosporidium spp., was performed. The amplicons obtained were purified and sequenced. PCR analysis revealed that 30 animals (15.3%) were positive for Cryptosporidium spp. There was no significant sex-dependent enrichment of Cryptosporidium occurrence (p > 0.05). Only 15 out of the 30 positive samples were successfully sequenced and their species determined, of which, 13 (86.7%) and 2 (13.3%) were C. meleagridis and C. galli, respectively. Herein, we present for the first time a molecular characterization of Cryptosporidium from feces of eared doves (Z. auriculata) and propose that these birds are a potential source of C. meleagridis infection in humans.

Resumo Cryptosporidium é um protozoário com uma grande variedade de hospedeiros, incluindo os seres humanos. No entanto, poucas espécies têm sido descritas em aves (Cryptosporidium meleagridis, C. baileyi, C. galli e C. avium). O objetivo do presente estudo foi investigar a ocorrência de Cryptosporidium spp. em fezes de pombas-de-bando (Zenaida auriculata), e realizar a caracterização molecular dos isolados. Um total de 196 animais de ambos os sexos foram capturados, eutanasiados e o conteúdo intestinal recolhido para extração de DNA. Após a extração, realizou-se uma nested-PCR (nPCR), que amplifica um fragmento do gene 18S rRNA do Cryptosporidium spp.. Os fragmentos obtidos foram purificados e encaminhados para sequenciamento. Os resultados da n-PCR revelaram 30 animais (15.3%) positivos para Cryptosporidium spp.. Quanto ao sexo dos animais não foram observadas diferenças estatísticas significativas (p > 0.05). Somente 15 de 30 amostras positivas foram sequenciadas com sucesso e as espécies determinadas, das quais, 13 (86.7%) e 2 (13.3%) foram C. meleagridis e C. galli, respectivamente. Esse é o primeiro estudo com caracterização molecular de Cryptosporidium de fezes de pombas-de-bando (Z. auriculata), e propõe serem esses animais potenciais fonte de infecção de C. meleagridis para humanos.

Morphological and molecular insights of a new species of trypanorhynchid cestode parasite, Nybelinia exostigmi, in the Narrowstripe cardinal fish Apogon exostigma / Abordagens morfológicas e moleculares de uma nova espécie de parasito cestóide tripanorrinquídeo, Nybelinia exostigmi, no peixe cardinal Narrowstripe Apogon exostigma

Abstract Forty specimens of the Narrowstripe cardinal fish Apogon exostigma were examined for gastrointestinal helminthes, and 62.5% were infected with a new trypanorhynchid larval cestode parasite. The morphology of its larval stage was studied based on light and scanning electron microscopy. The data revealed plerocercoid larvae characterized by a pyriform body lined with prominent microtriches; the acraspedote scolex had four overlapping bothridia; four tentacles protruded through the pars bothridialis; the armature of the tentacles was homeocanthous, homeomorphous, and consisted of falcate compact rose-thorn-shaped tentacular hooks; four oval-shaped bulbs in pars bulbosa; and short appendix at terminal end of the body. Molecular analysis of the 18S rRNA sequences verified the taxonomy of this parasite and supported its morphology. We discovered that there was a close identity (up to 87%) with alternative species obtained for comparison from GenBank. The data also showed that there were high blast scores and low divergence values between this parasite and other Tentaculariidae species. The phyletic analysis showed that parasite sequences in conjunction with existing data places this trypanorhynchid species among the Tentaculariidae. This species is deeply embedded within genus Nybelinia with close relationships to Nybelinia queenslandensis as a putative sister taxon.

Resumo Quarenta espécimes do peixe cardinal Apogon exostigma da Narrowstripe foram examinados para identificar helmintos gastrointestinais, destes 62,5% foram infectados com um novo parasito larval cestóide tripanorrinquídeo. A morfologia de seu estágio larval foi estudada na microscopia de luz e eletrônica de varredura. Os dados revelaram larvas plerocercoides caracterizadas por uma forma piriforme com um corpo revestido por microtrícinos proeminentes; o escolex acraspedótico tinha quatro sobreposições; quatro tentáculos se projetavam através da pars botridialis; a armadura dos tentáculos era homeocante, homeomorfa e consistia de ganchos tentaculares em forma de espinhos, em forma de falcão; quatro bulbos ovais em pars bulbosa; e apêndice curto na extremidade terminal do corpo. A análise molecular das sequências de RNAr 18S verificou a taxonomia desse parasita e apoiou sua morfologia. Descobrimos que havia uma identidade próxima (até 87%) com espécies alternativas obtidas para comparação do GenBank. Os dados também mostraram que houve altos escores de brusone e baixos valores de divergência entre este parasita e outras espécies de Tentaculariidae. A análise filética mostrou que as sequências de parasitas em conjunto com os dados existentes colocam esta espécie de tripanorimidídeo entre os Tentaculariidae. Esta espécie está profundamente enraizada no gênero Nybelinia, tendo relações próximas com Nybelinia queenslandensis como um putativo táxon irmão.

Surveillance of Giardia and Cryptosporidium in sewage from an urban area in Brazil / Monitoramento de Giardia e Cryptosporidium em esgoto de uma área urbana no Brasil

Abstract Cryptosporidium and Giardia are protozoan parasites that cause diarrhea in humans and animals. Molecular characterization of these pathogens in sewage may provide insight on their occurrence and prevalence in Brazil. This study aimed to investigate the presence of Giardia and Cryptosporidium in raw and treated sewage from Londrina, Paraná, Brazil. Samples were collected every two weeks during a year. Samples were concentrated, then DNA was extracted and subjected to a nested PCR targeting the Giardia 18S rRNA gene and the Cryptosporidium 18S rRNA gene. Species of Cryptosporidium were characterized by restriction fragment length polymorphism (RFLP). All raw sewage and 76% of the treated sewage were positive for Giardia; 84% of raw sewage samples and 8% of treated sewage were positive for Cryptosporidium. C. muris, C. hominis, C. baileyi, C. parvum and C. suis were detected in 100%, 19%, 9%, 9% and 4% of raw sewage, respectively. C. muris was the only species found in treated sewage. Multiple species of Cryptosporidium were present in 19.04% of the raw sewage. Treated sewage water can pose a threat to human health. The speciation of Cryptosporidium revealed the presence of non-common zoonotic species as C. suis and C. muris.

Resumo Cryptosporidium e Giardia são protozoários causadores de diarreia em animais e humanos. A caracterização molecular destes protozoários em esgoto pode prover dados ainda desconhecidos da ocorrência de espécies. O objetivo do presente estudo foi monitorar a ocorrência de Giardia e espécies de Cryptosporidium em esgoto bruto e tratado em uma estação de tratamento de esgoto (ETE) de Londrina, Paraná. Amostras de esgoto bruto e tratado foram coletadas no período de um ano, com periodicidade quinzenal. A ocorrência destes protozoários foi caracterizada por meio de concentração das amostras e posterior extração de DNA seguida de nested-PCR para amplificação de fragmentos dos genes 18S rRNA de Giardia e 18S rRNA de Cryptosporidium. A caracterização das espécies de Cryptosporidium foi realizada por meio de análise por polimorfismo de comprimento do fragmento de restrição (RFLP) dos produtos obtidos. Foram coletadas no total 25 amostras de cada, esgoto bruto e esgoto tratado. Para Giardia, todas as amostras de esgoto bruto e 76% das de esgoto tratado foram positivas. Cryptosporidium esteve presente em 84% das amostras de esgoto bruto e em 8% do tratado. No esgoto tratado foi encontrado apenas C. muris, já nas amostras de esgoto bruto foram encontradas cinco espécies: C. muris, C. hominis, C. baileyi, C. suis e C. parvum em 100%, 19%, 9%, 9% e 4%, respectivamente. A presença de espécies mistas foi observada em 19,04% das amostras. A presença de Giardia e Cryptosporidium em esgoto tratado pode pôr em risco a saúde humana. A discriminação de espécies de Cryptosporidium revelou a presença de espécies zoonóticas incomuns como C. suis e C. muris.

Detection molecular of Rangelia vitalii in dogs from Parana State, Southern Brazil / Detecção molecular de Rangelia vitalii em cães do Estado do Paraná, Sul do Brasil

Abstract Rangelia vitalii infects erythrocytes, leukocytes and endothelial cells of dogs. The present study aimed to report the molecular detection confirmed by sequencing of R. vitalii in the state of Paraná, as well as describe the clinical, hematological and biochemical alterations of the infected dogs. Three sick dogs from the metropolitan area of Curitiba, PR, Brazil, underwent a physical exam, and laboratory tests included hematology, biochemistry, polymerase chain reaction (PCR), and gene sequencing. Clinical signs included apathy, anorexia, and hemorrhage. Intra-erythrocytic and extracellular piroplasms were found on peripheral blood smears from all three dogs. Blood samples from these animals were positive for Babesia sp. by PCR targeting 18S rRNA. PCR products from all three dogs were sequenced, and BLAST analysis showed that the PCR-generated sequences were highly homologous with those of R. vitalii previously reported. Hematologic findings included severe anemia, shift of neutrophils to the regenerative left, and thrombocytopenia. Serum urea levels were increased in all three dogs, and direct bilirubin levels were elevated in one dog.

Resumo Rangelia vitalii infecta eritrócitos, leucócitos e células endoteliais de cães. O presente estudo objetivou relatar a detecção molecular confirmada por sequenciamento de R. vitalii no estado do Paraná e descrever as alterações clínicas, hematológicas e bioquímicas dos cães infectados. Três cães doentes da região metropolitana de Curitiba, PR, Brasil, foram submetidos a exame físico e exames laboratoriais que incluíram hematologia, bioquímica, reação em cadeia da polimerase (PCR) e sequenciamento genético. Os sinais clínicos incluíram apatia, anorexia e hemorragia. Piroplasmas intra-eritrocíticos e extracelulares foram encontrados em esfregaços de sangue periférico dos três cães. As amostras de sangue destes animais foram positivas para Babesia sp. pela PCR baseada no gene 18S rRNA. Os produtos de PCR dos três cães foram sequenciados e a análise de BLAST mostrou que as seqüências geradas por PCR eram altamente homólogas com as de R. vitalii previamente relatadas. Os achados hematológicos incluíram anemia grave, desvio de neutrófilos à esquerda regenerativo e trombocitopenia. Os níveis de uréia no soro aumentaram nos três cães, e os níveis de bilirrubina direta foram elevados em um cão.

Application of 18S rDNA Clone Library to Detect Diatom Population Diversity in Dianchi / 法医学杂志

Objective To detect the diatom population diversity in Dianchi by constructing a 18S rDNA clone library. Methods DNA from diatoms in 6 water samples of Dianchi was amplified with diatom 18S rDNA specific primer.The 18S rDNA clone library was constructed, and clones were randomly selected for sequence. Sequence alignment was performed by BLAST. The diatom population distribution in Dianchi was analyzed and the phylogenetic tree of diatom 18S rDNA in Dianchi waters was established with the MEGA v7.0.14 software. Results Two hundred and forty clones were sequenced, with 167 diatom sequences obtained, including 11 diatom species such as Stephanodiscus, Diatoma, and Melosira. There were certain differences in diatom population distribution among the 6 samples. Conclusion The population distribution of diatom species in Dianchi shows unique features and the sequence analysis of diatom 18S rDNA has a certain reference value to the inference of forensic drowning sites.

Prevalence of infection of biomphalaria glabrata by schistosoma mansoni and the risk of urban schistosomiasis mansoni in Salvador, Bahia, Brazil

Abstract INTRODUCTION: Biomphalaria glabrata is considered to be responsible for the incidence of schistosomiasis in Brazil. Therefore, surveillance of areas where schistosomiasis is prevalent is fundamental for public health planning. This study was aimed to evaluate B. glabrata populations in water bodies of the city of Salvador, determine their distribution, estimate the prevalence of Schistosoma mansoni infections, characterize shed cercariae, and identify transmission foci. METHODS: Malacological surveys were carried out in 17 water collections from Salvador. Snail species were identified based on shell and mantle characteristics. Snails were evaluated for S. mansoni infection by exposure to light and via real time polymerase chain reaction (qPCR) using S. mansoni-18S rRNA subunit specific primers. RESULTS: 1,403 B. glabrata were collected. Classical cercarial shedding indicated that 5 snails (0.4%) were positive for S. mansoni. A higher prevalence of infections was found in Horta de Saramandaia (5.5%) and Lagoa do IAT (1.9%). Non-Schistosoma larvae, such as Xiphidiocercaria, Strigeidae, Spirorchiidae and Clinostomidae, were observed in 3.2% of the snails. S. mansoni DNA was detected in 6.2% snails via qPCR. CONCLUSIONS: B. glabrata is widely distributed in Salvador, as indicated by 7 water collections associated with a risk of schistosomiasis transmission. To our knowledge, this is the first study to identify B. glabrata eliminating cercariae of Clinostomidae, Strigeidae, and Spirorchiidae in Salvador. We propose that qPCR may be employed in combination with classical cercarial shedding. Estimating S. mansoni prevalence in snails by only considering the results of light exposure method classical into account may underestimate the problem.

Blankaartia sinnamaryi (Trombidiformes: Trombiculidae) parasitizing birds in southeastern Brazil, with notes on Rickettsia detection / Blankaartia sinnamaryi (Trombidiformes: Trombiculidae) parasitando aves no sudeste do Brasil, e notas sobre a detecção de Rickettsia

Abstract The larvae of the family Trombiculidae are ectoparasites of vertebrates, including birds. The bite of some species can cause deep lesions and severe skin reactions in the host, these can lead to dermatitis, popularly known as trombiculiasis. A morphological study of chiggers collected on birds from the state of Minas Gerais in Southeastern Brazil discovered Blankaartia sinnamaryi-infestation on Passeriformes birds. Molecular studies of the disclosed the 18S rDNA sequences of the mite, and the detection of a Rickettsia sp. in this chigger mite species.

Resumo As larvas da família Trombiculidae são ectoparasitas de vertebrados, incluindo aves. A picada de algumas espécies pode causam lesões profundas e reações cutâneas graves no hospedeiro, estas podem levar a dermatites, popularmente conhecidas como trombiculíases. Por meio de um estudo morfológico dos espécimes coletados parasitando aves do estado de Minas Gerais, Sudeste do Brasil relatou a infestação por Blankaartia sinnamaryi em aves Passeriformes. Além disso, nós fornecemos sequências de rDNA 18S desses ácaros e a detecção de uma espécie de Rickettsia sp. nesta espécie de trombiculídeo.

Strongyloidiasis in humans: diagnostic efficacy of four conventional methods and real-time polymerase chain reaction

Abstract INTRODUCTION: Strongyloides stercoralis is an intestinal parasitic nematode that causes hyperinfection and/or a dissemination syndrome in hosts, which is often difficult to diagnose. This study aims to compare the diagnostic efficacy of four conventional methods used to diagnose strongyloidiasis with real-time polymerase chain reaction (qPCR) to detect S. stercoralis in fecal samples. METHODS: We analyzed 143 fecal samples collected from Colombian regions with varying degrees of risk for intestinal infections caused by S. stercoralis to assess the validity, performance, overall efficiency, and concordance of the qPCR using a direct stool test, modified Ritchie concentration technique, agar plate culture, and Harada-Mori technique as reference tests. RESULTS While four fecal samples were positive for S. stercoralis using conventional methods, 32 were positive via qPCR. The diagnostic sensitivity of the qPCR was 75% [95% confidence interval (CI): 20.07-100%], whereas its specificity, negative predictive value, negative likelihood ratio, and Youden's J index were 78.42% (95% CI: 71.22-85.62%), 99.09% (95% CI: 96.86-100%), 0.32 (95% CI: 0.06-1.74), and 0.53, respectively. In addition, the estimated kappa index between the qPCR and the conventional methods was 0.12 (95% CI: -0.020-0.26). CONCLUSIONS: The diagnostic sensitivity of qPCR to detect strongyloidiasis is analogous to that of conventional parasitology methods, with an additional advantage of being capable of identifying the parasite DNA at low sample concentrations.

Molecular Description of Macroorchis spinulosus (Digenea: Nanophyetidae) Based on ITS1 Sequences

We performed a molecular genetic study on the sequences of 18S ribosomal RNA (ITS1 region) gene in 4-day-old adult worms of Macroorchis spinulosus recovered in mice experimentally infected with metacercariae from crayfish in Jeollanam-do Province, Korea. The metacercariae were round, 180 µm in average diameter, encysted with 2 layers of thick walls, but the stylet on the oral sucker was not clearly seen. The adult flukes were oval shape, and 760-820 µm long and 320-450 µm wide, with anterolateral location of 2 large testes. The phylogenetic tree based on ITS1 sequences of 6 M. spinulosus samples showed their distinguished position from other trematode species in GenBank. The most closely resembled group was Paragonimus spp. which also take crayfish or crabs as the second intermediate host. The present study is the first molecular characterization of M. spinulosus and provided a basis for further phylogenetic studies to compare with other trematode fauna in Korea.

Subtype Distribution of Blastocystis in Thai-Myanmar Border, Thailand

Blastocystis sp. is a common zoonotic intestinal protozoa which has been classified into 17 subtypes (STs). A cross-sectional study was conducted to determine the prevalence and subtype distribution of Blastocystis in villagers living on the Thai-Myanmar border, where the risk of parasitic infection is high. A total of 207 stool samples were collected and DNA was extracted. PCR and sequencing using primers targeting small-subunit ribosomal RNA (SSU rRNA) gene were performed. The prevalence of Blastocystis infection was 37.2% (77/207). ST3 (19.8%; 41/207) was the predominant subtype, followed by ST1 (11.6%; 24/207), ST2 (5.3%; 11/207), and ST4 (0.5%; 1/207). A phylogenetic tree was reconstructed using the maximum likelihood (ML) method based on the Hasegawa-Kishino-Yano + G + I model. The percentage of bootstrapped trees in which the associated taxa clustered together was relatively high. Some sequences of Blastocystis positive samples (TK18, 39, 46, 71, and 90) were closely related to animals (pig and cattle) indicating zoonotic risks. Therefore, proper health education in parasitic prevention for the villagers should be promoted to improve their personal hygiene. Further longitudinal studies are required to monitor the prevalence of parasitic infections after providing health education and to investigate Blastocystis ST in animals living in these villages.

Ancient Mitochondrial DNA Analyses of Ascaris Eggs Discovered in Coprolites from Joseon Tomb

Analysis of ancient DNA (aDNA) extracted from Ascaris is very important for understanding the phylogenetic lineage of the parasite species. When aDNAs obtained from a Joseon tomb (SN2-19-1) coprolite in which Ascaris eggs were identified were amplified with primers for cytochrome b (cyt b) and 18S small subunit ribosomal RNA (18S rRNA) gene, the outcome exhibited Ascaris specific amplicon bands. By cloning, sequencing, and analysis of the amplified DNA, we obtained information valuable for comprehending genetic lineage of Ascaris prevalent among pre-modern Joseon peoples.