Phenotypic and molecular characterization of fluoroquinolone resistant Pseudomonas aeruginosa isolates in Palestine

Abstract Fluoroquinolones are important antimicrobial agents for the treatment of Pseudomonas infections. A total of 11 isolates of P. aeruginosa were collected from different clinical samples from different medical centers in the North West Bank-Palestine during 2017. In this study, resistance to fluoroquinolones and secretions of -lactamases were detected by phenotypic methods, while presence of -lactamase gene sequences and other virulence factors were detected by PCR technique. PCR product for gyrA, parC and parE genes were sequenced for further analyses. The phylogenetic analyses, population diversity indices and haplotypes determination were conducted using computer programs MEGA version 6, DnaSP 5.1001 and median-joining algorithm in the program Network 5, respectively. Results of this study showed that the MIC for ciprofloxacin and norfloxacin had a range of 32-256 µg/ml. In addition, all isolates carried either exoT or exoT and exoY genes, different -lactamase genes and 82% of these isolates harbored class 1 integrons. Analyses of the gyrA, parC and parE sequences were found to be polymorphic, had high haplotype diversity (0.945-0.982), low nucleotide diversity (0.01225-0.02001) and number of haplotypes were 9 for each gyrA and parE genes and 10 haplotypes for parC gene. The founder haplotypes being Hap-1 (18%), Hap-2 (27.3%) and Hap-6 (9.1%) for gyrA, parC and parE genes, respectively. Two of ParE haplotypes were detected as indel haplotypes. The Median-joining- (MJ) networks constructed from haplotypes of these genes showed a star-like expansion. The neutrality tests (Tajimas D test and Fus Fs test) for these genes showed negative values. Palestinian fluoroquinolone resistant P. aeruginosa strains showed high MIC level for fluoroquinolones, -lactamase producers, carried type III secretion exotoxin-encoding genes, most of them had integrase I gene and had high level of mutations in QRDR regions in gyrA, parC and parE genes. All these factors may play an important role in the invasiveness of these strains and make them difficult to treat. Isolation of these strains from different medical centers, indicate the need for a strict application of infection control measures in Medical centers in the North West Bank-Palestine that aim to reduce expense and damage caused by P. aeruginosa infections. Molecular analyses showed that Palestinian fluoroquinolone resistant P. aeruginosa haplotypes are not genetically differentiated; however, more mutations may exist in these strains.

Resumo Fluoroquinolonas são agentes antimicrobianos importantes para o tratamento de infecções por Pseudomonas. Um total de 11 bacilos isolados de P. aeruginosa foram coletados de diferentes amostras clínicas provenientes de diferentes centros médicos na Cisjordânia-Palestina durante o ano de 2017. Neste estudo, resistência a fluoroquinolonas e secreções de -lactamases foram detectadas por métodos fenotípicos, enquanto a presença de sequências do gene -lactamase e outros fatores de virulência foram detectados pela técnica de PCR (Proteína C-reativa). O produto de PCR para os genes gyrA, parC e parE foram sequenciados para análises posteriores. As análises filogenéticas, os índices de diversidade populacional e a determinação de haplótipos foram realizados utilizando os softwares MEGA versão 6, DnaSP 5.1001 e o algoritmo de junção de mediana do programa Network 5, respectivamente. Os resultados deste estudo mostraram que a MIC para ciprofloxacina e norfloxacina tinha um intervalo de 32-256 µg/ml. Além disso, todos os bacilos isolados carregavam genes exoT ou exoT e exoY, genes de -lactamase diferentes e 82% desses isolados continham integrons de classe 1. As análises das sequências gyrA, parC e parE foram consideradas polimórficas, com alta diversidade de haplótipos (0,945-0,982), baixa diversidade de nucleotídeos (0,01225-0,02001) e o número de haplótipos foi de 9 para cada gene de gyrA e parE e 10 haplótipos para o gene parC. Os haplótipos fundadores são Hap-1 (18%), Hap-2 (27,3%) e Hap-6 (9,1%) para os genes gyrA, parC e parE, respectivamente. Dois dos haplótipos parE foram detectados como haplótipos InDel. As redes Median-joining (MJ) construídas a partir de haplótipos desses genes mostraram uma expansão semelhante à de uma estrela. Os testes de neutralidade (teste D de Tajima e teste Fs de Fu) para esses genes apresentaram valores negativos. As cepas palestinas de P. aeruginosa resistentes a fluoroquinolonas mostraram alto nível de MIC para fluoroquinolonas, produtores de -lactamase, genes codificadores de exotoxina de secreção tipo III, a maioria deles tinha o gene integrase I e tinha alto nível de mutações nas regiões QRDR nos genes gyrA, parC e parE. Todos esses fatores podem desempenhar um papel importante na invasão dessas cepas e torná-las difíceis de tratar. O isolamento dessas cepas em diferentes centros médicos, indica a necessidade de uma aplicação estrita de medidas de controle de infecção em centros médicos da Cisjordânia-Palestina que visam reduzir despesas e danos causados por infecções por P. aeruginosa. As análises moleculares mostraram que os haplótipos de P. aeruginosa resistentes à fluoroquinolona palestina não são geneticamente diferenciados; no entanto, mais mutações podem existir nessas cepas.

Genetic structure analysis of Mauritia flexuosa natural population from the Lençóis Maranhenses region using microsatellite markers

Buriti (Mauritia flexuosa L.f), a palm tree native to South America and widely distributed in Brazil, displays significant ecological, economic and biotechnological importance. However, the disorderly extractivism and environmental degradation of its endemic areas are leading to reductions in, and/or extinction of the buriti palm tree, causing ecological imbalance with significant economic losses for rural communities and genetic diversity. Consequently, populational genetic diversity studies have become relevant as a strategy for conserving the species. Therefore, this study evaluated the genetic structure of 10 populations from the Lençóis Maranhenses region, in the state of Maranhão, Brazil, using microsatellite markers. Results indicated that eight pairs displayed a high level of polymorphism in the populations evaluated (98.5 %). The genetic diversity estimations allowed for identifying 220 alleles (average of 9.5 alleles/loci), and the heterozygosity averages observed (Ho) were lower than the heterozygosity expected (He) in the population (0.16 and 0.64) and loci levels (0.15 and 0.65), respectively. The Shannon Index (I) mean value of 1.36 indicated high diversity and genotypic richness in the populations evaluated, while the population index (FST) indicated a low value (0.05) and the fixation index pertaining to individuals indicated a high value (FIS = 0.79) exhibiting a moderate population distribution structure, and pointing to greater diversity between individuals. Based on these results, populations denominated as PA and PB presented a high genetic similarity (0.219), while populations denominated as PF and PJ exhibited more distant genetic characteristics (0.519). These results can be correlated based on prioritization of conservation of this non–domesticated species, influenced by environmental characteristics, suggesting that the genetic diversity found should be conserved in a germplasm bank, and subsequently exploited in breeding programs.

Morphological and molecular evidences of Ascaridia galli in migratory quail Coturnix coturnix japonica from Baluchistan Pakistan / Evidências morfológicas e moleculares de Ascaridia galli em codornas migratórias Coturnix coturnix japonica, do Baluchistão, Paquistão

The current study was conducted to examine the point prevalence of gastrointestinal parasites of migratory quails. Due to its economic importance, the control of ascaridiosis is critical. Migration of birds is considered to enhance the global spread and cross-species transmission of pathogens. The current study was aimed to detect A.galli in migratory quails, a potential contributory risk factor for transmission of this parasite to local birds. A total of 230 migratory quails were trapped using nets from migratory routes in Balochistan and examined under the compound microscope for the presence of A. galli. Conventionally, A. galli was identified by its morphology with the presence of three large lips and absence of posterior esophageal bulb. Results revealed that out of 230, 120 (52.17%) quails were positive for A. galli by targeting COX1 gene (533 bp) by using conventional PCR. Further, the amplicon was sequenced which showed 99% similarity with A. galli publically available in NCBI Gen Bank. Phylogenetic analysis of sequences of our isolated parasite indicated the close relationship with A.galli isolated from chickens. In conclusion migratory quails and other migratory birds may play a key role in spreading and transmission of these parasites and other pathogens to domestic chicken. Therefore, strict biosecurity measures should be adopted especially for commercial poultry farms.

O presente estudo foi conduzido para examinar a prevalência pontual de parasitas gastrointestinais de codornas migratórias. Devido à sua importância econômica, o controle da ascaridiose é fundamental. Considera-se que a migração de aves aumenta a disseminação global e a transmissão entre espécies de patógenos. O presente estudo teve como objetivo detectar A. galli em codornas migratórias, um potencial fator de risco contributivo para a transmissão desse parasita para aves locais. Um total de 230 codornas migratórias foi capturado, usando redes de rotas migratórias no Baluchistão e examinadas sob o microscópio composto para a presença de A. galli. Convencionalmente, o A. galli foi identificado por sua morfologia com a presença de três grandes lábios e ausência de bulbo esofágico posterior. Os resultados revelaram que de 230, 120 (52,17%) codornas foram positivas para A. galli por direcionamento do gene COX1 (533 pb) usando PCR convencional. Além disso, o amplicon foi sequenciado, que mostrou 99% de similaridade com A. galli publicamente disponível no NCBI Gen Bank. A análise filogenética das sequências do nosso parasita isolado indicou a estreita relação com A. galli isolado de galinhas. Em conclusão, codornas migratórias e outras aves migratórias podem desempenhar papel fundamental na disseminação e transmissão desses parasitas e outros patógenos para as galinhas domésticas. Portanto, medidas rigorosas de biossegurança devem ser adotadas, especialmente para granjas comerciais.

Phenotypic and molecular characterization of fluoroquinolone resistant Pseudomonas aeruginosa isolates in Palestine / Caracterização fenotípica e molecular de isolados de Pseudomonas aeruginosa resistentes a fluoroquinolonas na Palestina

Fluoroquinolones are important antimicrobial agents for the treatment of Pseudomonas infections. A total of 11 isolates of P. aeruginosa were collected from different clinical samples from different medical centers in the North West Bank-Palestine during 2017. In this study, resistance to fluoroquinolones and secretions of ß-lactamases were detected by phenotypic methods, while presence of ß-lactamase gene sequences and other virulence factors were detected by PCR technique. PCR product for gyrA, parC and parE genes were sequenced for further analyses. The phylogenetic analyses, population diversity indices and haplotypes determination were conducted using computer programs MEGA version 6, DnaSP 5.1001 and median-joining algorithm in the program Network 5, respectively. Results of this study showed that the MIC for ciprofloxacin and norfloxacin had a range of 32-256 µg/ml. In addition, all isolates carried either exoT or exoT and exoY genes, different ß-lactamase genes and 82% of these isolates harbored class 1 integrons. Analyses of the gyrA, parC and parE sequences were found to be polymorphic, had high haplotype diversity (0.945-0.982), low nucleotide diversity (0.01225-0.02001) and number of haplotypes were 9 for each gyrA and parE genes and 10 haplotypes for parC gene. The founder haplotypes being Hap-1 (18%), Hap-2 (27.3%) and Hap-6 (9.1%) for gyrA, parC and parE genes, respectively. Two of ParE haplotypes were detected as indel haplotypes. The Median-joining- (MJ) networks constructed from haplotypes of these genes showed a star-like expansion. The neutrality tests (Tajima's D test and Fu's Fs test) for these genes showed negative values. Palestinian fluoroquinolone resistant P. aeruginosa strains showed high MIC level for fluoroquinolones, ß-lactamase producers, carried type III secretion exotoxin-encoding genes, most of them had integrase I gene and had high level of mutations in QRDR regions in gyrA, parC and parE genes. All these factors may play an important role in the invasiveness of these strains and make them difficult to treat. Isolation of these strains from different medical centers, indicate the need for a strict application of infection control measures in Medical centers in the North West Bank-Palestine that aim to reduce expense and damage caused by P. aeruginosa infections. Molecular analyses showed that Palestinian fluoroquinolone resistant P. aeruginosa haplotypes are not genetically differentiated; however, more mutations may exist in these strains.

Fluoroquinolonas são agentes antimicrobianos importantes para o tratamento de infecções por Pseudomonas. Um total de 11 bacilos isolados de P. aeruginosa foram coletados de diferentes amostras clínicas provenientes de diferentes centros médicos na Cisjordânia-Palestina durante o ano de 2017. Neste estudo, resistência a fluoroquinolonas e secreções de ß-lactamases foram detectadas por métodos fenotípicos, enquanto a presença de sequências do gene ß-lactamase e outros fatores de virulência foram detectados pela técnica de PCR (Proteína C-reativa). O produto de PCR para os genes gyrA, parC e parE foram sequenciados para análises posteriores. As análises filogenéticas, os índices de diversidade populacional e a determinação de haplótipos foram realizados utilizando os softwares MEGA versão 6, DnaSP 5.1001 e o algoritmo de junção de mediana do programa Network 5, respectivamente. Os resultados deste estudo mostraram que a MIC para ciprofloxacina e norfloxacina tinha um intervalo de 32-256 µg/ml. Além disso, todos os bacilos isolados carregavam genes exoT ou exoT e exoY, genes de ß-lactamase diferentes e 82% desses isolados continham integrons de classe 1. As análises das sequências gyrA, parC e parE foram consideradas polimórficas, com alta diversidade de haplótipos (0,945-0,982), baixa diversidade de nucleotídeos (0,01225-0,02001) e o número de haplótipos foi de 9 para cada gene de gyrA e parE e 10 haplótipos para o gene parC. Os haplótipos fundadores são Hap-1 (18%), Hap-2 (27,3%) e Hap-6 (9,1%) para os genes gyrA, parC e parE, respectivamente. Dois dos haplótipos parE foram detectados como haplótipos InDel. As redes Median-joining (MJ) construídas a partir de haplótipos desses genes mostraram uma expansão semelhante à de uma estrela. Os testes de neutralidade (teste D de Tajima e teste Fs de Fu) para esses genes apresentaram valores negativos. As cepas palestinas de P. aeruginosa resistentes a fluoroquinolonas mostraram alto nível de MIC para fluoroquinolonas, produtores de ß-lactamase, genes codificadores de exotoxina de secreção tipo III, a maioria deles tinha o gene integrase I e tinha alto nível de mutações nas regiões QRDR nos genes gyrA, parC e parE. Todos esses fatores podem desempenhar um papel importante na invasão dessas cepas e torná-las difíceis de tratar. O isolamento dessas cepas em diferentes centros médicos, indica a necessidade de uma aplicação estrita de medidas de controle de infecção em centros médicos da Cisjordânia-Palestina que visam reduzir despesas e danos causados por infecções por P. aeruginosa. As análises moleculares mostraram que os haplótipos de P. aeruginosa resistentes à fluoroquinolona palestina não são geneticamente diferenciados; no entanto, mais mutações podem existir nessas cepas.

Screening of plant growth-promoting bacteria isolated from sugarcane / Seleção de bactérias promotoras de crescimento de plantas isoladas de cana-de-açúcar

Plant growth-promoting bacteria (PGPB) are known to establish positive relationships with plants. They act in favoring plant nutrition, production of phytohormones, control of pathogens and enhancement of stress tolerance. Thus, this study aimed to isolate bacteria from soil, rhizosphere, and root endosphere from sugarcane cultivated in the Southeastern of Brazil, to prospect strains with potential for plant growth promotion. The samples were plated in Nutrient Agar medium, and the morphologically distinct colonies were isolated and analyzed about indoleacetic acid production, phosphate solubilization and the growth control of the phytopathogenic fungus Fusarium verticillioides. A total of 219 isolates were obtained, of which 86 from soil, 67 from rhizosphere and 66 from sugarcane root endosphere. The strains that presented more than one mechanism of plant growth promotion were identified by the sequencing of 16S gene. Most species belonged to the genus Bacillus, which has strains already used in various biological products for the control of diseases in agriculture. Some Bacillus species isolated in our study have never been isolated from sugarcane, and others have been studied for the first time as plant growth promoters. The isolated strains constitute an important microbial bank to be explored to compose innovative products for agriculture.(AU)

Bactérias promotoras de crescimento de plantas são conhecidas por estabelecer relações positivas com as plantas. Atuam no favorecimento da nutrição das plantas, produção de fitohormônios, controle de patógenos e aumento da tolerância ao estresse. Desta forma, este estudo teve como objetivo isolar bactérias do solo, rizosfera e endosfera radicular de cana-de-açúcar, cultivada na região Sudeste do Brasil, para prospectar cepas com potencial para promoção de crescimento vegetal. As amostras foram semeadas em meio Ágar Nutriente, e as colônias morfologicamente distintas foram isoladas e analisadas quanto à produção de ácido indolacético, solubilização de fosfato e controle de crescimento do fungo fitopatogênico Fusarium verticillioides. Foram obtidos 219 isolados, sendo 86 do solo, 67 da rizosfera e 66 da endosfera da raiz da cana-de-açúcar. As cepas que apresentaram mais de um mecanismo de promoção do crescimento vegetal foram identificadas pelo sequenciamento do gene 16S. A maioria das espécies pertence ao gênero Bacillus, que possui linhagens utilizadas em diversos produtos biológicos para o controle de doenças na agricultura. Algumas espécies de Bacillus nunca foram isoladas da cana-deaçúcar e outras foram estudadas pela primeira vez como promotoras de crescimento de plantas. As cepas isoladas constituem um importante banco microbiano a ser explorado para a composição de produtos inovadores para a agricultura.(AU)

Phenotypic and molecular characterization of fluoroquinolone resistant Pseudomonas aeruginosa isolates in Palestine / Caracterização fenotípica e molecular de isolados de Pseudomonas aeruginosa resistentes a fluoroquinolonas na Palestina

Fluoroquinolones are important antimicrobial agents for the treatment of Pseudomonas infections. A total of 11 isolates of P. aeruginosa were collected from different clinical samples from different medical centers in the North West Bank-Palestine during 2017. In this study, resistance to fluoroquinolones and secretions of β-lactamases were detected by phenotypic methods, while presence of β-lactamase gene sequences and other virulence factors were detected by PCR technique. PCR product for gyrA, parC and parE genes were sequenced for further analyses. The phylogenetic analyses, population diversity indices and haplotypes determination were conducted using computer programs MEGA version 6, DnaSP 5.1001 and median-joining algorithm in the program Network 5, respectively. Results of this study showed that the MIC for ciprofloxacin and norfloxacin had a range of 32-256 µg/ml. In addition, all isolates carried either exoT or exoT and exoY genes, different β-lactamase genes and 82% of these isolates harbored class 1 integrons. Analyses of the gyrA, parC and parE sequences were found to be polymorphic, had high haplotype diversity (0.945-0.982), low nucleotide diversity (0.01225-0.02001) and number of haplotypes were 9 for each gyrA and parE genes and 10 haplotypes for parC gene. The founder haplotypes being Hap-1 (18%), Hap-2 (27.3%) and Hap-6 (9.1%) for gyrA, parC and parE genes, respectively. Two of ParE haplotypes were detected as indel haplotypes. The Median-joining- (MJ) networks constructed from haplotypes of these genes showed a star-like expansion. The neutrality tests (Tajima’s D test and Fu’s Fs test) for these genes showed negative values. Palestinian fluoroquinolone resistant P. aeruginosa strains showed high MIC level for fluoroquinolones, β-lactamase producers, carried type III secretion exotoxin-encoding genes, most of them [...].

Fluoroquinolonas são agentes antimicrobianos importantes para o tratamento de infecções por Pseudomonas. Um total de 11 bacilos isolados de P. aeruginosa foram coletados de diferentes amostras clínicas provenientes de diferentes centros médicos na Cisjordânia-Palestina durante o ano de 2017. Neste estudo, resistência a fluoroquinolonas e secreções de β-lactamases foram detectadas por métodos fenotípicos, enquanto a presença de sequências do gene β-lactamase e outros fatores de virulência foram detectados pela técnica de PCR (Proteína C-reativa). O produto de PCR para os genes gyrA, parC e parE foram sequenciados para análises posteriores. As análises filogenéticas, os índices de diversidade populacional e a determinação de haplótipos foram realizados utilizando os softwares MEGA versão 6, DnaSP 5.1001 e o algoritmo de junção de mediana do programa Network 5, respectivamente. Os resultados deste estudo mostraram que a MIC para ciprofloxacina e norfloxacina tinha um intervalo de 32-256 µg/ml. Além disso, todos os bacilos isolados carregavam genes exoT ou exoT e exoY, genes de β-lactamase diferentes e 82% desses isolados continham integrons de classe 1. As análises das sequências gyrA, parC e parE foram consideradas polimórficas, com alta diversidade de haplótipos (0,945-0,982), baixa diversidade de nucleotídeos (0,01225-0,02001) e o número de haplótipos foi de 9 para cada gene de gyrA e parE e 10 haplótipos para o gene parC. Os haplótipos fundadores são Hap-1 (18%), Hap-2 (27,3%) e Hap-6 (9,1%) para os genes gyrA, parC e parE, respectivamente. Dois dos haplótipos parE foram detectados como haplótipos InDel. As redes Median-joining (MJ) construídas a partir de haplótipos desses genes mostraram uma expansão semelhante à de uma estrela. Os testes de neutralidade (teste D de Tajima e teste Fs de Fu) para esses genes apresentaram valores negativos. As cepas palestinas de P. aeruginosa resistentes a fluoroquinolonas mostraram alto nível de MIC para [...].

Phenotypic and molecular characterization of fluoroquinolone resistant Pseudomonas aeruginosa isolates in Palestine / Caracterização fenotípica e molecular de isolados de Pseudomonas aeruginosa resistentes a fluoroquinolonas na Palestina

Fluoroquinolones are important antimicrobial agents for the treatment of Pseudomonas infections. A total of 11 isolates of P. aeruginosa were collected from different clinical samples from different medical centers in the North West Bank-Palestine during 2017. In this study, resistance to fluoroquinolones and secretions of β-lactamases were detected by phenotypic methods, while presence of β-lactamase gene sequences and other virulence factors were detected by PCR technique. PCR product for gyrA, parC and parE genes were sequenced for further analyses. The phylogenetic analyses, population diversity indices and haplotypes determination were conducted using computer programs MEGA version 6, DnaSP 5.1001 and median-joining algorithm in the program Network 5, respectively. Results of this study showed that the MIC for ciprofloxacin and norfloxacin had a range of 32-256 µg/ml. In addition, all isolates carried either exoT or exoT and exoY genes, different β-lactamase genes and 82% of these isolates harbored class 1 integrons. Analyses of the gyrA, parC and parE sequences were found to be polymorphic, had high haplotype diversity (0.945-0.982), low nucleotide diversity (0.01225-0.02001) and number of haplotypes were 9 for each gyrA and parE genes and 10 haplotypes for parC gene. The founder haplotypes being Hap-1 (18%), Hap-2 (27.3%) and Hap-6 (9.1%) for gyrA, parC and parE genes, respectively. Two of ParE haplotypes were detected as indel haplotypes. The Median-joining- (MJ) networks constructed from haplotypes of these genes showed a star-like expansion. The neutrality tests (Tajimas D test and Fus Fs test) for these genes showed negative values. Palestinian fluoroquinolone resistant P. aeruginosa strains showed high MIC level for fluoroquinolones, β-lactamase producers, carried type III secretion exotoxin-encoding genes, most of them [...].(AU)

Fluoroquinolonas são agentes antimicrobianos importantes para o tratamento de infecções por Pseudomonas. Um total de 11 bacilos isolados de P. aeruginosa foram coletados de diferentes amostras clínicas provenientes de diferentes centros médicos na Cisjordânia-Palestina durante o ano de 2017. Neste estudo, resistência a fluoroquinolonas e secreções de β-lactamases foram detectadas por métodos fenotípicos, enquanto a presença de sequências do gene β-lactamase e outros fatores de virulência foram detectados pela técnica de PCR (Proteína C-reativa). O produto de PCR para os genes gyrA, parC e parE foram sequenciados para análises posteriores. As análises filogenéticas, os índices de diversidade populacional e a determinação de haplótipos foram realizados utilizando os softwares MEGA versão 6, DnaSP 5.1001 e o algoritmo de junção de mediana do programa Network 5, respectivamente. Os resultados deste estudo mostraram que a MIC para ciprofloxacina e norfloxacina tinha um intervalo de 32-256 µg/ml. Além disso, todos os bacilos isolados carregavam genes exoT ou exoT e exoY, genes de β-lactamase diferentes e 82% desses isolados continham integrons de classe 1. As análises das sequências gyrA, parC e parE foram consideradas polimórficas, com alta diversidade de haplótipos (0,945-0,982), baixa diversidade de nucleotídeos (0,01225-0,02001) e o número de haplótipos foi de 9 para cada gene de gyrA e parE e 10 haplótipos para o gene parC. Os haplótipos fundadores são Hap-1 (18%), Hap-2 (27,3%) e Hap-6 (9,1%) para os genes gyrA, parC e parE, respectivamente. Dois dos haplótipos parE foram detectados como haplótipos InDel. As redes Median-joining (MJ) construídas a partir de haplótipos desses genes mostraram uma expansão semelhante à de uma estrela. Os testes de neutralidade (teste D de Tajima e teste Fs de Fu) para esses genes apresentaram valores negativos. As cepas palestinas de P. aeruginosa resistentes a fluoroquinolonas mostraram alto nível de MIC para [...].(AU)

Genetic diversity and population structure of endangered rosewood from the Peruvian Amazon using ISSR markers / Diversidad genética y estructura poblacional de palo de rosa en peligro de extinción de la Amazonía Peruana utilizando marcadores ISSR

Palo de rosa, Aniba rosaeodora es una especie en peligro de extinción en los bosques amazónicos. Sus rodales naturales se han agotado debido a la sobreexplotación para la industria cosmética. Este estudio tuvo como objetivo investigar la diversidad genética y estructura poblacional de 90 accesiones de palo de rosa de ocho localidades en la Amazonía Peruana utilizando 11 marcadores de Inter Secuencias Simples Repetidas (ISSR). Los marcadores ISSR produjeron una suma de 378 bandas, de las cuales 375 (99,2%) fueron polimórficas, con un valor promedio de contenido de información de polimorfismo (PIC) de 0,774. El promedio del número efectivo de alelos (Ne), índice informativo de Shannon (I), diversidad genética (He) y diversidad genética total (Ht) fueron 1,485; 0,294; 0,453 y 0,252; respectivamente. El análisis de varianza molecular (AMOVA) mostró la presencia de máxima variabilidad dentro de las poblaciones (88%). El algoritmo Structure, neighbor joining y análisis de coordenadas principales (PCoA) agruparon las 90 accesiones de palo de rosa en tres poblaciones principales (A, B y C). Los índices de diversidad a nivel interpoblacional revelaron una mayor diversidad genética en la población A, debido al mayor flujo de genes. El análisis de neighbor joining agrupó las poblaciones A y B, mientras la población C fué divergente a nivel interpoblacional. Concluimos que la población A refleja mayor diversidad genética y debería priorizarse para futuros planes de manejo y conservación.(AU)

Rosewood, Aniba rosaeodora is an endangered species in Amazon forests and its natural stands have been heavily depleted due to over-exploitation for the cosmetic industry. This study aimed to investigate the genetic diversity and population structure of 90 rosewood accessions from eight localities in the Peruvian Amazon through 11 Inter Simple Sequence Repeats (ISSR) primers. The ISSR primers produced a sum of 378 bands, of which 375 (99.2%) were polymorphic, with an average polymorphism information content (PIC) value of 0.774. The mean effective number of alleles (Ne), Shannon informative index (I), gene diversity (He) and total gene diversity (Ht) were 1.485, 0.294, 0.453 and 0.252, respectively. Analysis of molecular variance (AMOVA) showed the presence of maximum variability within populations (88%). The Structure algorithm, neighbor joining and principal coordinate analysis (PCoA) grouped the 90 rosewood accessions into three main populations (A, B and C). Diversity indices at the inter-population level revealed a greater genetic diversity in population A, due to higher gene flow. The neighbor-joining analysis grouped populations A and B, while population C was found to be divergent at the inter population level. We concluded that population A reflects higher genetic diversity and should be prioritized for future management and conservation plans.(AU)

Old tools as new support for on farm conservation of different types of maize

A germplasm collection should represent the diversity of the target species and the gene pools associated with it. However, it is critical to establish collection plans which ensure such representativeness. At times it is difficult to identify the best strategy for collecting domesticated species that are conserved in situ/on farm, since, in general, the magnitude of the diversity existing in a geographical area was hitherto unknown. The Diversity Census methodology was developed for previous diagnosis of the diversity of Zea mays subsp. mays L., conserved by farmers in two municipalities in the far western region of the state of Santa Catarina, southern Brazil. The Diversity Census database allowed for the identification of the best strategy to collect different types of maize landraces. Thus, tests were carried out using two methods described for Core Collections (Modified Random Sampling and Maximization) and a third statistical method for random sampling, stratified by farm area. The Maximization method enabled the capture of all the morphological variation of the traits evaluated in the Diversity Census from the smallest sample size. The relevance of this result is the feasibility of adapting the Core Collection strategy in order to plan more efficient expeditions to collect maize landraces conserved in microregions. Such planning allows for organizing the collection work efficiently, reducing costs, simplifying the work of characterization and helping to plan integrated strategies of in situ/on fam conservation.

Old tools as new support for on farm conservation of different types of maize

A germplasm collection should represent the diversity of the target species and the gene pools associated with it. However, it is critical to establish collection plans which ensure such representativeness. At times it is difficult to identify the best strategy for collecting domesticated species that are conserved in situ/on farm, since, in general, the magnitude of the diversity existing in a geographical area was hitherto unknown. The Diversity Census methodology was developed for previous diagnosis of the diversity of Zea mays subsp. mays L., conserved by farmers in two municipalities in the far western region of the state of Santa Catarina, southern Brazil. The Diversity Census database allowed for the identification of the best strategy to collect different types of maize landraces. Thus, tests were carried out using two methods described for Core Collections (Modified Random Sampling and Maximization) and a third statistical method for random sampling, stratified by farm area. The Maximization method enabled the capture of all the morphological variation of the traits evaluated in the Diversity Census from the smallest sample size. The relevance of this result is the feasibility of adapting the Core Collection strategy in order to plan more efficient expeditions to collect maize landraces conserved in microregions. Such planning allows for organizing the collection work efficiently, reducing costs, simplifying the work of characterization and helping to plan integrated strategies of in situ/on fam conservation.(AU)

Cytogenetic mapping of the ALMT (aluminum-activated malate transporter) gene in wheat genotypes

Presence of the ALMT1 (aluminum-activated malate transporter) gene confers resistance to aluminum toxicity in Triticum aestivum (common wheat). No resistant cultivars of Triticum turgidum ssp. Durum Desf. (durum wheat) have been registered in Brazil. The aim of this study was to map the ALMT1 through application of the FISH (fluorescence in situ hybridization) technique in five wheat genotypes, common and durum, from the Active Germplasm Bank (AGB) of the Instituto Agronômico (IAC): BH 1146, P19, P33, Anahuac, and IAC 1003. FISH-ALMT1 signals were registered in Anahuac (sensitive) chromosomes and in BH 1146, P19, and P33 (resistant) chromosomes. In the three resistant genotypes, a characteristic double FISH signal was found, located in different chromosomes of the complements: in BH 1146 in chromosome 5D, in P19 in 3B, and in P33 in 6B. This FISH - ALMT1 mapping allows for introgression of aluminum resistance in sensitive cultivars through breeding programs using introgression lines containing these carrier chromosomes.

Cytogenetic mapping of the ALMT (aluminum-activated malate transporter) gene in wheat genotypes

Presence of the ALMT1 (aluminum-activated malate transporter) gene confers resistance to aluminum toxicity in Triticum aestivum (common wheat). No resistant cultivars of Triticum turgidum ssp. Durum Desf. (durum wheat) have been registered in Brazil. The aim of this study was to map the ALMT1 through application of the FISH (fluorescence in situ hybridization) technique in five wheat genotypes, common and durum, from the Active Germplasm Bank (AGB) of the Instituto Agronômico (IAC): BH 1146, P19, P33, Anahuac, and IAC 1003. FISH-ALMT1 signals were registered in Anahuac (sensitive) chromosomes and in BH 1146, P19, and P33 (resistant) chromosomes. In the three resistant genotypes, a characteristic double FISH signal was found, located in different chromosomes of the complements: in BH 1146 in chromosome 5D, in P19 in 3B, and in P33 in 6B. This FISH - ALMT1 mapping allows for introgression of aluminum resistance in sensitive cultivars through breeding programs using introgression lines containing these carrier chromosomes.(AU)

Biotecnologias da reprodução animal: de Aristóteles à edição gênica / Biotechnologies of animal reproduction: from Aristotle to gene editing

Atualmente, com a rapidez e a facilidade da difusão da informação, os jovens acadêmicos têm percepção de que o conhecimento é um produto pronto, onde as características podem ser manipuladas e empregadas como um objeto oriundo de uma fábrica. A compreensão da realidade que nos cerca é um desejo desde os primórdios da presença do homem sobre a terra e os conceitos se modificam na medida em que o conhecimento avança. As tecnologias reprodutivas surgiram e evoluíram ao longo dos tempos, levando a aplicações com diferentes impactos na produção e seleção animal, com repercussões na saúde animal, na saúde humana, na preservação de espécies em extinção e em diferentes cenários da atividade humana. Este texto traça uma linha de tempo das ações em reprodução animal, sob a ótica dos autores, dando ênfase ao gradual desenvolvimento das biotécnicas como ferramenta auxiliar na rotina experimental e nos diversos sistemas de produção animal.(AU)

Today the rapid and ease information diffusion makes young academics have a perception that knowledge is a ready product, where characteristics could be manipulated and used as an object from a factory. The understanding of the reality that surrounds us is a desire from the earliest days of man's presence on earth and the concepts change as knowledge advances. Reproductive technologies have emerged and evolved over time, leading to applications with different impacts on animal production and selection, with repercussions on animal and human health, on the preservation of endangered species and on different scenarios of human activity. This text summarizes the evolution of thought, knowledge and actions in animal reproduction from the perspective of the authors, emphasizing the gradual development of biotechnology as an auxiliary tool in the experimental routine and in the various systems of animal production.(AU)

Biotecnologias da reprodução animal: de Aristóteles à edição gênica / Biotechnologies of animal reproduction: from Aristotle to gene editing

Atualmente, com a rapidez e a facilidade da difusão da informação, os jovens acadêmicos têm percepção de que o conhecimento é um produto pronto, onde as características podem ser manipuladas e empregadas como um objeto oriundo de uma fábrica. A compreensão da realidade que nos cerca é um desejo desde os primórdios da presença do homem sobre a terra e os conceitos se modificam na medida em que o conhecimento avança. As tecnologias reprodutivas surgiram e evoluíram ao longo dos tempos, levando a aplicações com diferentes impactos na produção e seleção animal, com repercussões na saúde animal, na saúde humana, na preservação de espécies em extinção e em diferentes cenários da atividade humana. Este texto traça uma linha de tempo das ações em reprodução animal, sob a ótica dos autores, dando ênfase ao gradual desenvolvimento das biotécnicas como ferramenta auxiliar na rotina experimental e nos diversos sistemas de produção animal.

Today the rapid and ease information diffusion makes young academics have a perception that knowledge is a ready product, where characteristics could be manipulated and used as an object from a factory. The understanding of the reality that surrounds us is a desire from the earliest days of man's presence on earth and the concepts change as knowledge advances. Reproductive technologies have emerged and evolved over time, leading to applications with different impacts on animal production and selection, with repercussions on animal and human health, on the preservation of endangered species and on different scenarios of human activity. This text summarizes the evolution of thought, knowledge and actions in animal reproduction from the perspective of the authors, emphasizing the gradual development of biotechnology as an auxiliary tool in the experimental routine and in the various systems of animal production.

Biochemical and physical kernel properties of a standard maize hybrid in different TopCross Blends

A pilot experiment was undertaken in order to examine high oil populations of maize (Zea mays L.) to be used as pollinators in TopCross blends with commercial ZP341 standard hybrid. Five high oil populations (HOPs) from the Maize Research Institute (MRI) gene bank were chosen for this research, according to their high grain oil content, synchrony between silking of ZP341 and anthesis of the populations and good agronomic performances in 2012. Selfing of ZP341 and HOPs, as well as crosses of ZP341 cmsS sterile × HOPs were carried out in 2013. Oil content, fatty acid composition, protein and tryptophan content, and physical characteristics of the obtained kernels were measured. Four HOPs showed significant positive influence on the oil content in the TopCrosses (TC), 16.85 g kg1 on average. Oleic acid, which is the principal monounsaturated fatty acid, was significantly lower in all HOPs and all TCs, while selfed ZP341 had almost twice the average value typical for standard maize. However, this decrease in TCs was in a narrow range from 1 % (in TC-3) to 5 % (in TC-4) and the oleic content of TCs was on average higher by 60 % compared to the typical standard maize. Different favorable and unfavorable significant changes were detected in fatty acid compositions, protein and tryptophan contents and physical kernel properties for each potential TC combination. Results indicate differences in gene effects present in different TC combinations and underscore the need to examine each potential TC blend by conducting similar simple experiments.

Biochemical and physical kernel properties of a standard maize hybrid in different TopCross Blends

A pilot experiment was undertaken in order to examine high oil populations of maize (Zea mays L.) to be used as pollinators in TopCross blends with commercial ZP341 standard hybrid. Five high oil populations (HOPs) from the Maize Research Institute (MRI) gene bank were chosen for this research, according to their high grain oil content, synchrony between silking of ZP341 and anthesis of the populations and good agronomic performances in 2012. Selfing of ZP341 and HOPs, as well as crosses of ZP341 cmsS sterile × HOPs were carried out in 2013. Oil content, fatty acid composition, protein and tryptophan content, and physical characteristics of the obtained kernels were measured. Four HOPs showed significant positive influence on the oil content in the TopCrosses (TC), 16.85 g kg1 on average. Oleic acid, which is the principal monounsaturated fatty acid, was significantly lower in all HOPs and all TCs, while selfed ZP341 had almost twice the average value typical for standard maize. However, this decrease in TCs was in a narrow range from 1 % (in TC-3) to 5 % (in TC-4) and the oleic content of TCs was on average higher by 60 % compared to the typical standard maize. Different favorable and unfavorable significant changes were detected in fatty acid compositions, protein and tryptophan contents and physical kernel properties for each potential TC combination. Results indicate differences in gene effects present in different TC combinations and underscore the need to examine each potential TC blend by conducting similar simple experiments.(AU)

CÉLULAS TRONCO MESENQUIMAIS CANINAS CULTIVADAS COM DESFERROXAMINE (DFO) E INTERFERON GAMMA, MIMETIZANDO CONDIÇÕES DE HIPÓXIA E INFLAMAÇÃO

As células tronco mesenquimais (MSC) tem se destacado como candidatas ao uso em terapias regenerativas. Porém, foi demonstrado que nem todas as condições clínicas podem ser favorecidas pela presença das MSCs, já que suas propriedades são influenciadas pelas condições do microambiente onde serão aplicadas, que normalmente estão acompanhadas da presença de fatores inflamatórios e isquêmicos. Devido a isto, este estudo teve por objetivo avaliar os efeitos in vitro da adição de DFO e/ou ao IFN- no meio de cultivo de células tronco mesenquimais (MSCs do inglês Mesenchymal Stem Cells) de tecido adiposo canino (cAT-MSCs). Foram utilizadas MSCs originadas de 6 cadelas e pertencentes a um banco celular em terceira passagem. As amostras foram divididas em 4 grupos, controle (MSCs em condições normais de cultivo em meio de manutenção), grupo DFO (50 µmol de DFO no meio de manutenção), grupo IFN- (50 ng/mL de IFN- no meio de manutenção) e grupo IFN-/DFO (50 µmol de DFO e 50 ng/mL IFN- no meio de manutenção). O teste de proliferação foi feito por 144 horas de cultivo e, viabilidade, apoptose e expressão gênica foram avaliadas após 48 horas de cultivo. Na expressão genica foram analisados transcritos relacionados com apoptose (BCL2, CASP8 e CASP9), sobrevivência celular (DKC1, HDAC1 e DNMT1), imunomodulação (HGF, IDO, PGE2 e IL-6), proliferação e diferenciação (FGF2), angiogênese (MMP2 e VEGFA), reguladores do ciclo celular (Cyclin-D1 e TP53) e regulador celular de resposta adaptativa a hipóxia (HIF1-). As cAT-MSCs mostraram durante e ao final do cultivo morfologia fibroblastóide e aderência ao plástico. No entanto, os grupos tratados apresentaram maior distanciamento entre as células aderidas. Após 144 horas de cultivo, todos os grupos tiveram comportamento similar quanto a curva de proliferação celular, com diminuição da concentração nas primeiras 48 horas, seguido por uma fase de manutenção e finalizando com um ligeiro crescimento. A expressão gênica revelou que as cAT-MSCs tratadas com IFN- apresentaram um incremento na expressão do gene pró-apoptótico Casp9 quando comparado com o grupo IFN-/DFO; o gene FGF2 importante para os processos de proliferação e diferenciação celular foi aumentado no grupo IFN- quando comparado aos outros grupos tratados; os genes DKC1 e TP53 envolvidos com a sobrevida celular e supressão tumoral respectivamente, tiveram um incremento no grupo IFN- quando comparado com o grupo DFO; a expressão do gene relacionado com os processos de angiogênese, o VEGFA, foi maior nos grupos onde o DFO foi usado. Concluímos que condicionar as cAT-MSCs com a citocina pro-inflamatória IFN- estimulou um aumento na sobrevivência, proliferação celular e angiogênese mediada por MMP2. Já a mimetização de hipóxia, levou a uma diminuição da expressão de genes relacionados a estas propriedades associadas a um estímulo para a angiogênese dependente de VEGF. Apesar disso, os genes relacionados a imunomodulação não foram influenciados pelas condições de cultivo. Os resultados obtidos indicam que as propriedades de indução da regeneração tecidual foram mais afetadas pelas condições de cultivo, em comparação às propriedades imunomodulados das cAT-MSC

Mesenchymal stem cells (MSC) have been highlighted as candidates for use in regenerative therapies. However, it has been shown that not all clinical conditions can be favored by the presence of MSCs, as their properties are influenced by the conditions of the microenvironment where it will be applied, conditions that are usually accompanied by the presence of inflammatory and ischemic factors. So, this study aimed to evaluate the in vitro effects of the addition of DFO and/or IFN- in the culture medium of mesenchymal stem cells (MSCs Mesenchymal Stem Cells) from canine adipose tissue (cAT-MSCs). MSCs from 6 bitches and belonging to a cell bank in the third passage were used. Samples were divided in 4 groups: Control (MSCs under standard culture conditions in maintenance medium); DFO group (50 µM of DFO in maintenance medium), IFN- Group (50 ng/mL of IFN- in maintenance medium) and IFN-/DFO group (50 µM of DFO and 50 ng/mL IFN- in maintenance medium). Proliferation test was performed for 144 hours of culture and viability, apoptosis and gene expression were evaluated after 48 hours of culture. In gene expression, transcripts related to apoptosis (BCL2, CASP8 and CASP9), cell survival (DKC1, HDAC1 and DNMT1), immunomodulation (HGF, IDO, PGE2 and IL-6), proliferation and differentiation (FGF2), angiogenesis (MMP2 and VEGFA), cell cycle regulators (Cyclin-D1 and TP53) and cellular regulator of adaptive response to hypoxia (HIF1-) were analyzed. The cAT-MSCs showed fibroblastoid morphology and plastic adherence during and at the end of the culture. However, the treated groups showed greater distance between adhered cells. After 144 hours of culture, all groups had a similar behavior regarding the cell proliferation curve, with a decrease in concentration in the first 48 hours, followed by a maintenance phase and ending with a slight growth. Gene expression revealed that cAT-MSCs treated with IFN- showed an increase in the expression of the pro-apoptotic Casp9 gene when compared to the IFN-/DFO group; the FGF2 gene important for cell proliferation and differentiation processes was increased in the IFN- group when compared to the other treated groups; the DKC1 and TP53 genes involved with cell survival and tumor suppression respectively, had an increase in the IFN- group when compared to the DFO group; the expression of the gene related to the angiogenesis processes, VEGFA, was higher in the groups where DFO was used. We conclude that conditioning cAT-MSCs with the pro-inflammatory cytokine IFN- stimulated an increase in survival, cell proliferation and MMP2-mediated angiogenesis. The mimicry of hypoxia, on the other hand, led to a decrease in the expression of genes related to these properties associated with a stimulus for VEGF-dependent angiogenesis. Despite this, genes related to immunomodulation were not influenced by culture conditions. The results obtained indicate that tissue regeneration induction properties were more affected by culture conditions, compared to the immunomodulated properties of cAT-MSC.

Canine heartworm disease in a brazilian non-endemic area / Dirofilariose canina em uma aréa não endêmica brasileira / Dirofilariosis canina en una zona no endémica brasileña

Canine Heartworm Disease (CHD) is a mosquito-borne disease caused by Dirofilaria immitis. In this study, two mature adult-senior dogs from a non-endemic area to CHD presented clinical signs suggestive to the disease. The first one presented skin lesions, loss of appetite, weakness, pale mucosa membrane, and hyperthermia, whereas the second one presented severe ascites, anorexia and exercise intolerance, lateral decumbency, and marked heart murmurs. Both presented tachypnea, thrombocytopenia, leukocytosis, and microfilaremia. Multiplex-PCR (COI gene) resulted positive to D. immitis research in both cases, confirmed by sequencing, with 98% homology to D. immitis (Gen Bank accession n.AJ537512-1). In addition, both animals have never had any prophylactic treatment to CHD, and no reports about traveling to coastal areas. This study reported two unusual cases of D. immitis infection in non-endemic area from Brazil.(AU)

A dirofilariose canina (CHD) é uma doença transmitida por mosquitos e causada por Dirofilaria immitis. No presente estudo, dois cães de idade adulta a idoso de área não endêmica apresentaram sinais clínicos sugestivos da doença. O primeiro apresentou lesões de pele, perda de apetite, fraqueza, mucosas pálidas e hipertermia, enquanto o segundo apresentou severo quadro de ascite, anorexia e intolerância ao exercício, decúbito lateral, e murmúrios cardíacos acentuados. Ambos apresentaram taquipneia, trombocitopenia, leucocitose e microfilaremia. A pesquisa por D. immitis pela multiplex-PCR (COI gene) resultou positiva em ambos os casos, confirmada pelo sequenciamento, com 98% de homologia com D. immitis (Gen Bank n. AJ537512-1). Nenhum dos animais havia sido submetido a tratamento profilático para CHD e não havia relatos de viagens para regiões litorâneas. Assim, o presente estudo reporta dois casos raros de infecção por D. immitis em área brasileira não endêmica para a doença.(AU)

La dirofilariosis canina (CHD) es una enfermedad transmitida por mosquitos y causada por Dirofilaria immitis. En este estudio, dos perros de edad adulta a anciano, de área no endémica presentaron signos clínicos de la enfermedad. El primero presentó lesiones en la piel, pérdida del apetito, debilidad, palidez de mucosas e hipertermia, mientras el otro presentó severa ascitis, anorexia e intolerancia al ejercicio, decúbito lateral, y soplos cardíacos acentuados. Ambos presentaron taquipnea, trombocitopenia, leucocitosis y microfilaremia. La investigación de D. immitis por multiplex-PCR (gen COI) resultó positivo en ambos casos, confirmados por la técnica de secuenciación, con 98% de homología con D. immitis (Gen Bank n.AJ537512-1). Ninguno de los animales había sido sometido al tratamiento profiláctico para CHD, y sin relatos de viajes a regiones costeras. El presente estudio reporta dos casos raros de la infección por D. immitis en zona no endémica de Brasil.(AU)

Canine heartworm disease in a brazilian non-endemic area / Dirofilariose canina em uma aréa não endêmica brasileira / Dirofilariosis canina en una zona no endémica brasileña

Canine Heartworm Disease (CHD) is a mosquito-borne disease caused by Dirofilaria immitis. In this study, two mature adult-senior dogs from a non-endemic area to CHD presented clinical signs suggestive to the disease. The first one presented skin lesions, loss of appetite, weakness, pale mucosa membrane, and hyperthermia, whereas the second one presented severe ascites, anorexia and exercise intolerance, lateral decumbency, and marked heart murmurs. Both presented tachypnea, thrombocytopenia, leukocytosis, and microfilaremia. Multiplex-PCR (COI gene) resulted positive to D. immitis research in both cases, confirmed by sequencing, with 98% homology to D. immitis (Gen Bank accession n.AJ537512-1). In addition, both animals have never had any prophylactic treatment to CHD, and no reports about traveling to coastal areas. This study reported two unusual cases of D. immitis infection in non-endemic area from Brazil.

A dirofilariose canina (CHD) é uma doença transmitida por mosquitos e causada por Dirofilaria immitis. No presente estudo, dois cães de idade adulta a idoso de área não endêmica apresentaram sinais clínicos sugestivos da doença. O primeiro apresentou lesões de pele, perda de apetite, fraqueza, mucosas pálidas e hipertermia, enquanto o segundo apresentou severo quadro de ascite, anorexia e intolerância ao exercício, decúbito lateral, e murmúrios cardíacos acentuados. Ambos apresentaram taquipneia, trombocitopenia, leucocitose e microfilaremia. A pesquisa por D. immitis pela multiplex-PCR (COI gene) resultou positiva em ambos os casos, confirmada pelo sequenciamento, com 98% de homologia com D. immitis (Gen Bank n. AJ537512-1). Nenhum dos animais havia sido submetido a tratamento profilático para CHD e não havia relatos de viagens para regiões litorâneas. Assim, o presente estudo reporta dois casos raros de infecção por D. immitis em área brasileira não endêmica para a doença.

La dirofilariosis canina (CHD) es una enfermedad transmitida por mosquitos y causada por Dirofilaria immitis. En este estudio, dos perros de edad adulta a anciano, de área no endémica presentaron signos clínicos de la enfermedad. El primero presentó lesiones en la piel, pérdida del apetito, debilidad, palidez de mucosas e hipertermia, mientras el otro presentó severa ascitis, anorexia e intolerancia al ejercicio, decúbito lateral, y soplos cardíacos acentuados. Ambos presentaron taquipnea, trombocitopenia, leucocitosis y microfilaremia. La investigación de D. immitis por multiplex-PCR (gen COI) resultó positivo en ambos casos, confirmados por la técnica de secuenciación, con 98% de homología con D. immitis (Gen Bank n.AJ537512-1). Ninguno de los animales había sido sometido al tratamiento profiláctico para CHD, y sin relatos de viajes a regiones costeras. El presente estudio reporta dos casos raros de la infección por D. immitis en zona no endémica de Brasil.

Análise de parentesco e endogamia usando informações de pedigree e painel multiplex em um banco genético de tilápias

A aquicultura é uma atividade em franca expansão, no qual a tilápia (Oreochromis niloticus) é considerada uma espécie de extrema relevância para o setor, configurando como uma das mais cultivadas no mundo. Apesar do potencial produtivo da tilápia, se faz necessária a utilização de ferramentas, como marcadores do tipo SSR, capazes de auxiliar no diagnóstico de animais endogâmicos nos sistemas de cultivo, o que acarretaria na perda de ganho genético. Sendo assim, o estudo objetivou identificar marcadores SSR 4-mer, 5-mer e 6-mer potencialmente amplificáveis (PALs) para tilápia, assim como desenvolver um painel multiplex para análise de parentesco de animais oriundos de programas de seleção, visando a redução de inferências errôneas dos genótipos. De forma concomitante, foi aferida a estrutura populacional de um programa de seleção da espécie, utilizando análise de pedigree. Foi utilizado o genoma completo da tilápia depositado no banco de dados Genbank para mineração de SSRs candidatos e PALs. Utilizando a mesma metodologia, foi desenvolvido um painel composto apenas de marcadores 4-mer, ao qual foram analisados posteriormente por eletroforese capilar. Quanto à análise de pedigree, foram avaliados 12990 tilápias oriundos de 287 famílias distribuídos em 5 gerações de um banco genético privado. Foram sintetizados 10 marcadores altamente polimórficos para a construção do painel, onde os alelos foram amplificados sem bandas stutter e facilmente interpretados. Ademais, foram identificados 1689 novos marcadores (PALs), podendo os mesmos serem inseridos em futuros estudos de parentesco da espécie. Por sua vez, o programa de seleção analisado apresentou endogamia e parentesco dentro do aceitável, havendo a possibilidade de formação de novas gerações sem haver depressão endogâmica.

Aquaculture is a rapidly expanding activity, in which tilapia (Oreochromis niloticus) is considered an extremely relevant species for the sector, being one of the most cultivated in the world. Despite the productive potential of tilapia, it is necessary to use tools, such as SSR markers, able to assist in the diagnosis of inbred animals in the culture systems, which would result in the loss of gain in the tilapia production. Therefore, this study aimed to identify potentially amplifiable 4-mer, 5-mer and 6-mer SSR markers (PALs) for tilapia, as well as to develop a multiplex panel for kinship analysis of animals from selection programs, aiming to reduce erroneous inferences of genotypes. Simultaneously, the population structure of a selection program of the species, using pedigree analysis. We used the complete tilapia genome available in the GenBank database was used for mining candidate SSRs and PALs. Using the same approach, a panel composed only of 4-mer markers, which were further analyzed by capillary electrophoresis. As for pedigree analysis, 12990 tilapia from 287 families distributed in 5 generations of a private gene bank. Ten highly polymorphic markers were synthesized for the construction the panel, where the alleles were amplified without stutter bands and easily interpreted. Furthermore, 1689 new markers (PALs) were identified, which can be to be included in future studies of relatedness of the species. In turn, the selection program analyzed showed endogamy and kinship within acceptable levels, with the possibility of developing new generations without inbreeding depression.