Morphology and molecular phylogeny of trypanorhynchid metacestodes infecting commercial fish of the Mediterranean Sea / Morfologia e filogenia molecular das metacestodes de tripanorhynchid que infectam os peixes comerciais do Mar Mediterrâneo

Members of the order Trypanorhyncha are cestode parasites that are frequently found infecting the muscles of several marine fish species, affecting fish health, and resulting in consumers' rejection of fish. Fifty-two specimens of marine fish were freshly caught throughout the year 2020 from boat landing sites at the Alexandria coast along the Mediterranean Sea in Egypt, including the grey trigger fish Balistes carolinensis (F: Balistidae); the mottled grouper Mycteroperca rubra (F: Serranidae) and the common sole Solea vulgaris (F: Soleidae). Blastocysts were isolated and ruptured; the generated pleurocerci were described morphologically and morphometrically by light and scanning electron microscopy. Also, multiple-sequence alignment was performed, and a phylogenetic tree was constructed following maximum likelihood analysis of the 18s and 28s ribosomal RNA sequences of the recovered worms. Thirty fish were infected; the infection was recorded as blastocysts embedded in fish flesh. Three different parasitic species were recovered and classified morphologically as Gymnorhynchus isuri, Pseudotobothrium dipsacum and Heteronybelinia estigmena. The taxonomic position of these parasites was justified by molecular analysis of their 18s and 28s rRNAs, which revealed high percentages of homology with species recovered from the GenBank. The accession numbers ON157059, ON139663 and ON139662 were respectively assigned to the recovered parasites after their deposition in GenBank. The results obtained from the molecular analyses confirmed the morphological records of the recovered parasites. Since metacestodes are found in the musculature of infected fish specimens, it is necessary to remove these areas in the commercialization of fish.

Os membros da ordem Trypanorhyncha são parasitas de cestóides que são freqüentemente encontrados infectando os músculos de várias espécies de peixes marinhos, afetando a saúde dos peixes e resultando na rejeição do peixe por parte dos consumidores. Cinqüenta e dois espécimes de peixes marinhos foram capturados recentemente durante todo o ano de 2020 nos locais de desembarque de barcos na costa de Alexandria ao longo do Mar Mediterrâneo, no Egito, incluindo o peixe de gatilho cinzento Balistes carolinensis (F: Balistidae); a garoupa mosqueada Mycteroperca rubra (F: Serranidae) e o linguado comum Solea vulgaris (F: Soleidae). Os blastocistos foram isolados e rompidos; os pleurocistos gerados foram descritos morfologicamente e morfometricamente por microscopia eletrônica de luz e varredura. Além disso, foi realizado o alinhamento de sequências múltiplas e uma árvore filogenética foi construída seguindo a análise de máxima probabilidade das sequências de RNA ribossômico de 18s e 28s dos vermes recuperados. Trinta peixes foram infectados; a infecção foi registrada como blastocistos embutidos na carne do peixe. Três espécies diferentes de parasitas foram recuperadas e classificadas morfologicamente como Gymnorhynchus isuri, Pseudotobothrium dipsacum e Heteronybelinia estigmena. A posição taxonômica desses parasitas foi justificada pela análise molecular de seus rRNAs de 18 e 28 anos, que revelou altas porcentagens de homologia com espécies recuperadas do GenBank. Os números de acesso ON157059, ON139663 e ON139662 foram respectivamente atribuídos aos parasitas recuperados após sua deposição no GenBank. Os resultados obtidos a partir das análises moleculares confirmaram os registros morfológicos dos parasitas recuperados. Como as metacestodes são encontradas na musculatura dos espécimes de peixes infectados, é necessário remover estas áreas na comercialização dos peixes.

Isolation of Mycoplasma spp. from Geese with Pneumonia and Identification of Microbial Isolates via Molecular Methods

This study aimed to investigate Mycoplasma species in the lungs of 500 geese with pneumonia from the Kars region (Turkey) via cultural and molecular methods. The samples were cultured on Freys Broth and Agar media. To identify Mycoplasma species a Growth Inhibition Test was used. The identification was continued with species-specific PCR and sequence analysis which provide amplification of the genes dnaX, pcrA, rpoB, and the sequence of the 16S rRNA gene, respectively. In addition, Mycoplasma gallisepticum and Mycoplasma synoviae from pneumonic lung samples were directly analyzed via Multiplex Real-time PCR. As a result, 51 Mycoplasma strains were isolated and 32 were identified as Mycoplasma anatis, 9 as Mycoplasma anseris, 5 as Mycoplasma cloacale and 3 as Mycoplasma anserisalpingitis. Two Mycoplasma isolates that could not be identified were grouped in the same branch as a result of 16S RNA sequencing and their nearest neighbour was found to be Mycoplasma sp. 2045 (GenBankNo.MK615061.1). M. gallisepticum DNA was detected in 3 pneumonic lung samples and M. gallisepticum/M. synoviae DNAs were found simultaneously in 1 sample. While some Mycoplasma species identified in this study consolidated their place as pneumonic agents, some increased their potential to become a pneumonic agent when compared with cases caused by well-recognized Mycoplasma strains. Two isolates were identified as -Mycoplasma spp. as their 16S rRNA gene sequence identity levels scored below the threshold of 98.7% for species demarcation and still need to be defined whether they are possible representatives of a novel Mycoplasma species.(AU)

Isolation of Mycoplasma spp. from Geese with Pneumonia and Identification of Microbial Isolates via Molecular Methods

This study aimed to investigate Mycoplasma species in the lungs of 500 geese with pneumonia from the Kars region (Turkey) via cultural and molecular methods. The samples were cultured on Frey’s Broth and Agar media. To identify Mycoplasma species a Growth Inhibition Test was used. The identification was continued with species-specific PCR and sequence analysis which provide amplification of the genes dnaX, pcrA, rpoB, and the sequence of the 16S rRNA gene, respectively. In addition, Mycoplasma gallisepticum and Mycoplasma synoviae from pneumonic lung samples were directly analyzed via Multiplex Real-time PCR. As a result, 51 Mycoplasma strains were isolated and 32 were identified as Mycoplasma anatis, 9 as Mycoplasma anseris, 5 as Mycoplasma cloacale and 3 as Mycoplasma anserisalpingitis. Two Mycoplasma isolates that could not be identified were grouped in the same branch as a result of 16S RNA sequencing and their nearest neighbour was found to be Mycoplasma sp. 2045 (GenBankNo.MK615061.1). M. gallisepticum DNA was detected in 3 pneumonic lung samples and M. gallisepticum/M. synoviae DNAs were found simultaneously in 1 sample. While some Mycoplasma species identified in this study consolidated their place as pneumonic agents, some increased their potential to become a pneumonic agent when compared with cases caused by well-recognized Mycoplasma strains. Two isolates were identified as -Mycoplasma spp. as their 16S rRNA gene sequence identity levels scored below the threshold of 98.7% for species demarcation and still need to be defined whether they are possible representatives of a novel Mycoplasma species.

Phylogenetic analysis: basic concepts and its use as a tool for virology and molecular epidemiology / Análise filogenética: conceitos básicos e suas utilizações como ferramenta para virologia e epidemiologia molecular

Background: Phylogenetic analyses are an essential part in the exploratory assessment of nucleic acid and amino acid sequences. Particularly in virology, they are able to delineate the evolution and epidemiology of disease etiologic agents and/or the evolutionary path of their hosts. The objective of this review is to help researchers who want to use phylogenetic analyses as a tool in virology and molecular epidemiology studies, presenting the most commonly used methodologies, describing the importance of the different techniques, their peculiar vocabulary and some examples of their use in virology. Review: This article starts presenting basic concepts of molecular epidemiology and molecular evolution, emphasizing their relevance in the context of viral infectious diseases. It presents a session on the vocabulary relevant to the subject, bringing readers to a minimum level of knowledge needed throughout this literature review. Within its main subject, the text explains what a molecular phylogenetic analysis is, starting from a multiple alignment of nucleotide or amino acid sequences. The different software used to perform multiple alignments may apply different algorithms. To build a phylogeny based on amino acid or nucleotide sequences it is necessary to produce a data matrix based on a model for nucleotide or amino acid replacement, also called evolutionary model. [...](AU)

Phylogenetic analysis: basic concepts and its use as a tool for virology and molecular epidemiology / Análise filogenética: conceitos básicos e suas utilizações como ferramenta para virologia e epidemiologia molecular

Background: Phylogenetic analyses are an essential part in the exploratory assessment of nucleic acid and amino acid sequences. Particularly in virology, they are able to delineate the evolution and epidemiology of disease etiologic agents and/or the evolutionary path of their hosts. The objective of this review is to help researchers who want to use phylogenetic analyses as a tool in virology and molecular epidemiology studies, presenting the most commonly used methodologies, describing the importance of the different techniques, their peculiar vocabulary and some examples of their use in virology. Review: This article starts presenting basic concepts of molecular epidemiology and molecular evolution, emphasizing their relevance in the context of viral infectious diseases. It presents a session on the vocabulary relevant to the subject, bringing readers to a minimum level of knowledge needed throughout this literature review. Within its main subject, the text explains what a molecular phylogenetic analysis is, starting from a multiple alignment of nucleotide or amino acid sequences. The different software used to perform multiple alignments may apply different algorithms. To build a phylogeny based on amino acid or nucleotide sequences it is necessary to produce a data matrix based on a model for nucleotide or amino acid replacement, also called evolutionary model. [...]

Identificação de Mycobacterium bovis em carcaças de bovinos abatidos no estado da Bahia, Brasil, por métodos bacteriológico e molecular / Identification of Mycobacterium bovis in carcasses of cattle slaughtered in Bahia state, Brazil by bacteriological and molecular methods

O objetivo do presente trabalho foi utilizar métodos bacteriológicos e moleculares para a identificação do Mycobacterium bovis em lesões observadas em carcaças de bovinos durante a inspeção post mortem de rotina em matadouros-frigoríficos com serviço de inspeção oficial. Foram acompanhados o abate e a inspeção de 825.394 bovinos, sadios, ao exame ante mortem pelo serviço de inspeção oficial em 10 matadouros-frigoríficos do estado da Bahia, entre abril de 2009 e abril de 2012. Cento e oitenta bovinos apresentaram lesões sugestivas de tuberculose e outras linfadenites, as quais foram avaliadas quanto à presença de Mycobacterium bovis por exame bacteriológico e pela PCR multiplex. A maioria das lesões estava localizada em linfonodos do trato respiratório e 71% eram provenientes de bovinos machos com até 32 meses de idade. No isolamento bacteriano, 13,9% (25/180) das amostras apresentavam colônias pequenas, de superfície granular e de coloração creme-amareladas, em meio de cultura Stonebrink-Leslie, e o crescimento médio foi de 34 dias. Todos os esfregaços dos isolados evidenciaram BAAR, e, pela PCR multiplex, 56% (14/25) dos isolados foram identificados como M. bovis. A associação entre exame post mortem, bacteriológico e PCR multiplex permitiu a identificação do agente de forma rápida e em regiões com status sanitário de baixa prevalência, demonstrando ser importante para a detecção dos focos de tuberculose bovina e o auxílio nos programas de controle e erradicação da tuberculose.(AU)

The aim of the present study was to perform bacteriological and molecular methods for identification of Mycobacterium bovis in lesions derived from bovine carcasses detected during routine post-mortem examination in officially inspected slaughterhouses. We checked the slaughter and inspection of 825,394 bovines, health upon ante-mortem examination, by the official service in 10 slaughterhouses of Bahia state from April, 2009 to April 2012. Lesions suggestive of tuberculosis were collected from 180 bovines and further evaluated by bacteriology and multiplex PCR. The majority of lesions were located in the respiratory tract lymph nodes and 71% were from male bovines up to 32 months old. 13.9% of samples presented small, granular and creamy-yellowish colonies after being cultured in Stonebrink-Leslie with an average growth time of 34 days. All smears from the isolated samples were Acid Fast Bacilli (AFB) and among them 56% were identified by mPCR as M. bovis. Thus, the association between post-mortem examination, culture and multiplex PCR allowed the bacillus identification in a reduced time and in regions of low prevalence, pointing out its importance for bovine tuberculosis detection and as a supportive tool for the tuberculosis control and eradication program.(AU)

Expressão gênica de lectinas em caprinos e ovinos infectados por nematoides gastrintestinais / Gene expression of lectins in goats and sheep infected with gastrointestinal nematodes

As colectinas e galectinas são proteínas da família das lectinas de mamíferos que possuem a capacidade de reconhecer padrões moleculares associados a patógenos. Estudos em bovinos demonstraram a indução dessas proteínas na infecção por nematódeos gastrintestinais (NGIs), sugerindo que estas moléculas podem ter papel relevante no controle de parasitos. A detecção e caracterização de marcadores homólogos às colectinas bovinas no genoma caprino e ovino e a detecção de sua expressão no tecido abomasal são dados inéditos nestas espécies. A expressão dos genes de colectinas e galectinas em tecido do abomaso de caprinos com diferentes graus de infecção parasitária foi acompanhada pela ativação de genes IL-4 e IFN- ?. Em caprinos não foi observada diferença na expressão entre os grupos resistentes e suscetíveis aos NGIs, ao contrário dos ovinos, que apresentaram uma maior expressão dos genes COLBOV, SPA e GAL14 em animais com maior grau de infecção. Assim, evidenciamos a participação das lectinas na resposta imune contra nematoides gastrintestinais, sugerindo que as colectinas e galectinas podem estar envolvidas no reconhecimento de padrões moleculares associadas aos helmintos. (AU)

The galectins and collectins are proteins lectins family from mammals that arecapable of recognizing pathogen-associated molecular patterns. Studies in animals have demonstrated the induction of these proteins in infection with gastrointestinal nematodes (NGIs), suggesting that these molecules may play a relevant role in controlling parasites. The detection and characterization of markers homol ogous to bovine collectins in goat and sheep genome and detection of its expression in tissue abomasal are unpublished data on these species. Gene expression of galectins and collectins in tissue abomasum of goats with different levels of parasitic infection was accompanied by the activation of genes IL-4 andIFN-γ. In goats there was no difference in expression be tween the resistant and susceptible groups to NGIs, unlike sheep, which had a higher expression of genes COLBOV, SPA and GAL14 in animals with greater infection. Thus, we noted the involvement of lectins in theimmune response against gastrointestinal nematodes, suggesting that the collectins and galectins may be involved in the recognition of molecular patterns associated with helminths. (AU)

Expressão gênica de lectinas em caprinos e ovinos infectados por nematoides gastrintestinais / Gene expression of lectins in goats and sheep infected with gastrointestinal nematodes

As colectinas e galectinas são proteínas da família das lectinas de mamíferos que possuem a capacidade de reconhecer padrões moleculares associados a patógenos. Estudos em bovinos demonstraram a indução dessas proteínas na infecção por nematódeos gastrintestinais (NGIs), sugerindo que estas moléculas podem ter papel relevante no controle de parasitos. A detecção e caracterização de marcadores homólogos às colectinas bovinas no genoma caprino e ovino e a detecção de sua expressão no tecido abomasal são dados inéditos nestas espécies. A expressão dos genes de colectinas e galectinas em tecido do abomaso de caprinos com diferentes graus de infecção parasitária foi acompanhada pela ativação de genes IL-4 e IFN- ?. Em caprinos não foi observada diferença na expressão entre os grupos resistentes e suscetíveis aos NGIs, ao contrário dos ovinos, que apresentaram uma maior expressão dos genes COLBOV, SPA e GAL14 em animais com maior grau de infecção. Assim, evidenciamos a participação das lectinas na resposta imune contra nematoides gastrintestinais, sugerindo que as colectinas e galectinas podem estar envolvidas no reconhecimento de padrões moleculares associadas aos helmintos.

The galectins and collectins are proteins lectins family from mammals that arecapable of recognizing pathogen-associated molecular patterns. Studies in animals have demonstrated the induction of these proteins in infection with gastrointestinal nematodes (NGIs), suggesting that these molecules may play a relevant role in controlling parasites. The detection and characterization of markers homol ogous to bovine collectins in goat and sheep genome and detection of its expression in tissue abomasal are unpublished data on these species. Gene expression of galectins and collectins in tissue abomasum of goats with different levels of parasitic infection was accompanied by the activation of genes IL-4 andIFN-γ. In goats there was no difference in expression be tween the resistant and susceptible groups to NGIs, unlike sheep, which had a higher expression of genes COLBOV, SPA and GAL14 in animals with greater infection. Thus, we noted the involvement of lectins in theimmune response against gastrointestinal nematodes, suggesting that the collectins and galectins may be involved in the recognition of molecular patterns associated with helminths.