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Canine otitis externa is a disease that affects the external acoustic meatus of about 20% of dogs at some point in life, without predilection for race, age, or sex. It is a multifactorial disease whose etiology involves several microorganisms, detaching Staphylococcus aureus. Antimicrobials are the basis for treating this illness; however, due to the increase in antimicrobial resistance, conventional drugs have become ineffective, requiring the search for alternative therapies. In this context, essential oils (EOs) have great therapeutic potential due to their broad antimicrobial action. This study aimed to evaluate the minimum inhibitory concentration (MIC) in order to measure the MIC50 and MIC90 of gentamicin and EOs of Eugenia caryophyllata, Thymus vulgaris, Cymbopogon winterianus, Cymbopogon citratus, and Cinnamomum cassia against 62 Staphylococcus aureus strains isolated from the external acoustic meatus of dogs. All EOs showed antibacterial action against the studied microorganisms, and their MIC50 and MIC90 were as follows: Eugenia caryophyllata, 2.42 mg mL-1 and 7.25 mg mL-1; Thymus vulgaris, 9.51 mg mL-1 and 22.94 mg mL-1; Cymbopogon winterianus, 26.78 mg mL-1 and 157.79 mg mL-1; Cinnamomum cassia and Cymbopogon citratus, lower than 16.48 and 27.81 mg mL-1, with the same MIC for all isolates. The MIC50 and MIC90 found for gentamicin were 1ug mL-1and 8 ug mL-1. The MIC range found to antibiotic in this assay was 0.5 ug mL-1to 128 ug mL-1, and the isolates were classified as susceptible [48 strains (77.41%) - MIC range of 0.5-4.0 ug mL-1], intermediate [eight strains (12.90%) - (MIC = 8.0 ug mL-1], or resistant [six strains (9.68%) - MIC bigger, smaller 16 ug mL-1]. The results, according to the in vitro assays, showed that resistance to gentamicin, one of the antimicrobials most commonly used to treat canine otitis, is present in the Staphylococcus aureus population evaluated. Additionally, the tested EOs have great potential for therapeutic use, however future studies should be carried out to evaluate their in vivo efficacy. A otite canina externa é uma doença que afeta o meato acústico externo de até 20% dos cães em algum período de suas vidas, sem predileção por raça, idade ou sexo. Trata-se de uma doença multifatorial que tem Staphylococcus aureus como um dos principais agentes etiológicos. Antimicrobianos constituem a base para o tratamento desta enfermidade, entretanto, devido ao incremento da resistência antimicrobiana, os fármacos convencionais tem se tornado pouco eficazes, o que requer a busca por terapias alternativas. Neste contexto, os óleos essenciais (OE) apresentam grande potencial terapêutico devido a sua ampla ação antimicrobiana. Este trabalho visou detectar a concentração inibitória mínima (CIM), a partir da qual foram obtidos os índices de MIC50 e MIC90 para a gentamicina e para os OE de Eugenia caryophyllata, Thymus vulgaris, Cymbopogon winterianus, Cymbopogon citratus e Cinnamomum cassia contra 62 Staphylococcus aureus isolados do meato acústico externo de cães otopatas. Todos os OE mostraram ação antibacteriana contra os microrganismos estudados e foram detectadas as seguintes CIM50 e CIM90: Eugenia caryophyllata, 2,42 mg mL-1e 7,45 mg mL-1; Thymus vulgaris, 9,51 mg mL-1 e 22,94 mg mL-1; Cymbopogon winterianus, 26,78 mg mL-1 e 157,79 mg mL-1; Cinnamomum cassia e Cymbopogon citratus CM menores que 16,48 e 27,81 mg mL-1, respectivamente já que estes apresentaram CIM única para todos os isolados testados. Para a gentamicina, foram obtidas CIM50 e CIM90 1ug mL-1 e 8 ug/m, respectivamente. Ademais, a faixa de CIM encontrada para o antibiótico variou de 0,5 a 128 ug mL-1, e os isolados foram classificados em susceptíveis [48 isolados (77,41%) - CIM na faixa de 0,5-4 ug mL-1], intermediários [8 isolados (12,90%) - (CIM = 8ug mL-1] e resistentes [6 isolados (9,68%) - CIM maior, menor 16]. Os resultados encontrados mostraram que os isolados de micro-organismos testados tem potencial para desenvolver resistência antimicrobiana a terapêutica tradicional e que os OE, de acordo com os resultados in vitro, apresentam potencial de uso terapêutico, entretanto futuros estudos devem ser realizados para avaliar a eficácia dos OE in vivo.

Rhodnius prolixus is a Triatominae insect species and a primary vector of Chagas disease. The genome of R. prolixus has been recently sequenced and partially assembled, but few transcriptome analyses have been performed to date. In this study, we describe the stage-specific transcriptomes obtained from previtellogenic stages of oogenesis and from mature eggs. By analyzing ~ 228 million paired-end RNA-Seq reads, we significantly improved the current genome annotations for 9206 genes. We provide extended 5′ and 3′ UTRs, complete Open Reading Frames, and alternative transcript variants. Strikingly, using a combination of genome-guided and de novo transcriptome assembly we found more than two thousand novel genes, thus increasing the number of genes in R. prolixus from 15,738 to 17,864. We used the improved transcriptome to investigate stage-specific gene expression profiles during R. prolixus oogenesis. Our data reveal that 11,127 genes are expressed in the early previtellogenic stage of oogenesis and their transcripts are deposited in the developing egg including key factors regulating germline development, genome integrity, and the maternal-zygotic transition. In addition, GO term analyses show that transcripts encoding components of the steroid hormone receptor pathway, cytoskeleton, and intracellular signaling are abundant in the mature eggs, where they likely control early embryonic development upon fertilization. Our results significantly improve the R. prolixus genome and transcriptome and provide novel insight into oogenesis and early embryogenesis in this medically relevant insect.

The Triatominae subfamily (Reduviidae) harbors some hematophagous insect species that have been firmly connected to the transmission of Trypanosoma cruzi, the causative agent of Chagas disease. Triatomines not only host and transmit trypanosomatids, but also coexist with a variety of symbiotic microorganisms that generally reside in the insect’s intestinal flora. The microbiome has profound effects on the physiology, immunity, fitness and survival of animals and plants. The interaction between triatomines and bacteria has been investigated to some extent and has revealed important bacteria symbionts. In contrast, the range of viral species that can infect triatomine insects is almost completely unknown. In some cases, genomic and metatranscriptomic approaches have uncovered sequences related to possible viral genomes, but, to date, only eight positive single-strand RNA viruses, namely Triatoma virus and Rhodnius prolixus viruses 1 - 7 have been investigated in more detail. Here, we review the literature available on triatomine viruses and the viruses-insect host relationship. The lack of broader metagenomic and metatranscriptomic studies in these medically relevant insects underscores the importance of expanding our knowledge of the triatomine virome both for surveillance purposes as well as to possibly harness their potential for insect vector population control strategies.

Triatomine assassin bugs comprise hematophagous insect vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Although the microbiome of these species has been investigated to some extent, only one virus infecting Triatoma infestans has been identified to date. Here, we describe for the first time seven (+) single-strand RNA viruses (RpV1-7) infecting Rhodnius prolixus, a primary vector of Chagas disease in Central and South America. We show that the RpVs belong to the Iflaviridae, Permutotetraviridae and Solemoviridae and are vertically transmitted from the mothers to the progeny via transovarial transmission. Consistent with this, all the RpVs, except RpV2 that is related to the entomopathogenic Slow bee paralysis virus, established persistent infections in our R. prolixus colony. Furthermore, we show that R. prolixus ovaries express 22-nucleotide viral siRNAs (vsiRNAs), but not viral piRNAs, that originate from the processing of dsRNA intermediates during viral replication of the RpVs. Interestingly, the permutotetraviruses and sobemoviruses display shared pools of vsiRNAs that might provide the basis for a cross-immunity system. The vsiRNAs are maternally deposited in the eggs, where they likely contribute to reduce the viral load and protect the developing embryos. Our results unveil for the first time a complex core virome in R. prolixus and begin to shed light on the RNAi-based antiviral defenses in triatomines., Author summary Rhodnius prolixus is a triatomine insect and a primary vector of Trypanosoma cruzi, the etiologic agent of Chagas disease, in Central and South America. Despite the medical relevance, very little is known about the viruses that infect these so-called assassin bugs. In this study, we show for the first time that triatomines can support the concomitant infection of a variety of RNA viruses belonging to distantly related viral families. Remarkably, we show that the viruses are vertically transmitted from the mothers to the progeny via transovarial transmission. The detection of 22-nucleotide viral small interfering RNAs in mature eggs strongly suggests that RNAi mechanisms contribute to reduce the viral load during oogenesis and embryogenesis in R. prolixus, thus safeguarding the development of embryos and nymphs. In agreement with these findings, all the viruses, except one, could establish persistent infections in our colony. Our results substantially expand the knowledge of the virus complexity in triatomine species. This viral toolkit might be harnessed to develop novel insect population control strategies to reduce the diffusion of Chagas disease.

Rhodnius prolixusis a Triatominae insect species and a primary vector of Chagas disease. The genome ofR. prolixushas been recently sequenced and partially assembled, but few transcriptome analyses have been performed to date. In this study, we describe the stage-specific transcriptomes obtained from previtellogenic stages of oogenesis and from mature eggs. By analyzing ~228 million paired-end RNA-Seq reads, we significantly improved the current genome annotations for 9,206 genes. We provide extended 5’ and 3’ UTRs, complete Open Reading Frames, and alternative transcript variants. Strikingly, using a combination of genome-guided andde novotranscriptome assembly we found more than two thousand novel genes, thus increasing the number of genes inR. prolixusfrom 15,738 to 17,864. We used the improved transcriptome to investigate stage-specific gene expression profiles duringR. prolixusoogenesis. Our data reveal that 11,127 genes are expressed in the early previtellogenic stage of oogenesis and their transcripts are deposited in the developing egg including key factors regulating germline development, genome integrity, and the maternal-zygotic transition. In addition, GO term analyses show that transcripts encoding components of the steroid hormone receptor pathway, cytoskeleton, and intracellular signaling are abundant in the mature eggs, where they likely control early embryonic development upon fertilization. Our results significantly improve theR. prolixusgenome and transcriptome and provide novel insight into oogenesis and early embryogenesis in this medically relevant insect.

Triatomine assassin bugs comprise hematophagous insect vectors ofTrypanosoma cruzi, the causative agent of the Chagas disease. Although the microbiome of these species has been investigated to some extent, only one virus infectingTriatoma infestanshas been identified to date. Here, we describe for the first time seven (+) single-strand RNA viruses (RpV1-7) infectingRhodnius prolixus, a primary vector of the Chagas disease in Central and South America. We show that the RpVs belong to the Picorna-Calici, Permutotetra and Luteo-Sobemo clades and are vertically transmitted from the mothers to the progeny via transovarial transmission. Consistent with this, all the RpVs, except RpV2 that is related to the entomopathogenic Slow bee paralysis virus, established persistent infections in ourR. prolixuscolony. Furthermore, we show thatR. prolixusovaries express 22-nucleotide viral siRNAs (vsiRNAs), but not viral piRNAs, that originate from the processing of dsRNA intermediates during viral replication of the RpVs. Interestingly, the Permutotetra and Luteo-Sobemo viruses display shared pools of visRNAs that might provide the basis for a cross-immunity system. The vsiRNAs are maternally deposited in the eggs, where they likely contribute to reduce the viral load and protect the developing embryos. Our results unveil for the first time a complex core virome inR. prolixusand begin to shed light on the RNAi-based antiviral defenses in triatomines.Author summaryRhodnius prolixusis a triatomine insect and a primary vector ofTrypanosoma cruzi, the etiologic agent of the Chagas disease, in Central and South America. Despite the medical relevance, very little is known about the viruses that infect these so-called assassin bugs. In this study, we show for the first time that triatomines can support the concomitant infection of a variety of RNA viruses belonging to distantly related viral families. Remarkably, we show that the viruses are vertically transmitted from the mothers to the progeny via transovarial transmission. The detection of 22-nucleotide viral small interfering RNAs in mature eggs strongly suggests that RNAi mechanisms contribute to reduce the viral load during oogenesis and embryogenesis inR. prolixus, thus safeguarding the development of embryos and nymphs. In agreement with these findings, all the viruses, except one, could establish persistent infections in our colony. Our results substantially expand the knowledge of the virus complexity in triatomine species. This viral toolkit might be harnessed to develop novel insect population control strategies to reduce the diffusion of the Chagas disease.

Streptococcus agalactiae is one of the most common pathogens leading to mastitis in dairy herds worldwide; consequently, the pathogen causes major economic losses for affected farmers. In this study, multilocus sequence typing (MLST), genotypic capsular typing by multiplex polymerase chain reaction (PCR), and virulence gene detection were performed to address the molecular epidemiology of 59 bovine (mastitis) S. agalactiae isolates from 36 dairy farms located in the largest milk-producing mesoregions in Brazil (Minas Gerais, São Paulo, Paraná, and Pernambuco). We screened for the virulence genes bac, bca, bibA, cfb, hylB, fbsA, fbsB, PI-1, PI-2a, and PI-2b, which are associated with adhesion, invasion, tissue damage, and/or immune evasion. Furthermore, five capsular types were identified (Ia, Ib, II, III, and IV), and a few isolates were classified as non-typeable (NT). MLST revealed the following eight sequence types (STs): ST-61, ST-67, ST-103, ST-146, ST-226, ST-314, and ST-570, which were clustered in five clonal complexes (CC64, CC67, CC103, CC17, and CC314), and one singleton, ST-91. Among the virulence genes screened in this study, PI-2b, fbsB, cfb, and hylB appear to be the most important during mastitis development in cattle. Collectively, these results establish the molecular epidemiology of S. agalactiae isolated from cows in Brazilian herds. We believe that the data presented here provide a foundation for future research aimed at developing and implementing new preventative and treatment options for mastitis caused by S. agalactiae.

325 Background: Our aim was to investigate outcome of patients (pts) treated with AC or NC for organ-confined UC, or with NC for clinical lymph node involvement (cN+). Methods: We retrospectively analyzed 482 pts treated in 22 French hospitals between 2000 and 2013 in 3 settings: 226 pts with radical cystectomy (RC) and AC (AC group), 193 cN0 (NC group), and 63 cN+ (cN+ group) pts received NC before a planned RC for cT2-T4, M0 UC. The primary outcome was overall survival (OS). Cancer-related survival (CRS) was also evaluated. Multivariate Cox proportional hazards regression was used to estimate adjusted hazard ratios with 95% confidence interval. Results: Median age was 62, 61.7, and 62.1 in AC, NC, and cN+, respectively. Median time between diagnosis and first treatment (RC or NC) was 2.1, 1.4 and 1.6 months, respectively (p

4550 Background: The benefit of NC on survival in UC has been shown with SD and is most evident in patients (pts) who achieve a pathological complete response (ypT0). In the last decade, physicians...

TO THE EDITOR: In a recent letter, Alexandrescu et al reported thoughtful data to address the occurrence of erythrocytosis after treatment with tyrosine kinase inhibitors (TKIs) sorafenib and sunitinib for metastatic cancers. In addition, it has been previously reported that patients with von Hippel-Lindau (VHL) disease treated with semaxanib, a TKI targeting vascular endothelial growth factor receptor 2 (VEGFR-2) and cKit, may experience a supra-additive increase in hematocrit level. Experimental studies showing hepatic erythropoietin (EPO) synthesis related to VEGF blockage could explain this iatrogenic polycythemia. We report a case of polycythemia in a patient treated for metastatic renal cell carcinoma with axitinib, another TKI targeting VEGFR-2. Our findings were the opposite of those in the report by Alexandrescu et al. A 52-year-old man, with no significant past medical history, underwent a right radical nephrectomy for clear renal cell carcinoma. In October 2002, a partial response was achieved with interferon-alfa treatment for lung metastases. In March 2004, the lung metastases progressed in number and size, and the patient subsequently was enrolled onto an axitinib phase II trial. Oral axitinib was administered continuously at the fixed dose of 5 mg twice daily. Minor adverse effects were reported, including diarrhea and asthenia. During the initiation of the second cycle (week 5), we observed an increase in hematocrit, which was confirmed during the subsequent cycles. An isotopic measurement of RBCs confirmed the diagnosis of polycythemia. Treatment was continued for 36 months, with a documented complete response by the third cycle. However, repeated therapeutic phlebotomies for polycythemia remained necessary. There was no alternative cause of polycythemia, including the absence of clinical evidence of VHL disease. In addition, no somatic VHL mutation in the tumor sample was found in polymerase chain reaction analysis. Endogenous EPO blood levels and VEGF-A levels were monitored, and results are listed in Table 1. Tam et al demonstrated that erythropoiesis is regulated by endogenous VEGF. In Tam’s preclinical study, high-grade VEGF blockage induced the repression of adult hepatic EPO production. The absence of VEGFR-2 expression on hepatocytes suggests the participation of liver sinusoidal endothelial cells with homeostatic VEGFR-2– dependant paracrine signaling. In our observation, axitinib treatment was associated with a paradoxical VEGF increase, as reported both in vivo and in a cohort of patients with metastatic gastrointestinal stromal tumors treated with sunitinib, another TKI with VEGFR-2 inhibition. However, we speculate that axitinib mimics VEGF starvation in organs (such as the liver) through VEGFR-2 blockade, and leads to hepatic EPO production. Finally, we cannot exclude an axitinib interaction with the hypoxiainducible factor pathway, which induces nitric oxide release, cutaneous vascular flow modification, and increased systemic EPO expression. Further investigation in clinical trials is warranted to determine the utility of EPO blood level as a marker of VEGF/ VEGFR modulator activity.