Search

The emergence of drug-resistant strains of the parasite

Objectives To characterize the clonal spread of carbapenem-resistant Klebsiella pneumoniae and Escherichia coli isolates between different healthcare institutions in Catalonia, Spain. Methods Antimicrobial susceptibility was tested by disc diffusion. MICs were determined by gradient diffusion or broth microdilution. Carbapenemase production was confirmed by lateral flow. PCR and Sanger sequencing were used to identify the allelic variants of resistance genes. Clonality studies were performed by PFGE and MLST. Plasmid typing, conjugation assays, S1-PFGE plus Southern blotting and MinION Oxford Nanopore sequencing were used to characterize resistance plasmids. Results Twenty-nine carbapenem-resistant isolates recovered from three healthcare institutions between January and November 2016 were included: 14 K. pneumoniae isolates from a tertiary hospital in the south of Catalonia (hospital A); 2 K. pneumoniae isolates from a nearby healthcare centre; and 12 K. pneumoniae isolates and 1 E. coli isolate from a tertiary hospital in Barcelona (hospital B). The majority of isolates were resistant to all antimicrobial agents, except colistin, and all were NDM producers. PFGE identified a major K. pneumoniae clone (n = 27) belonging to ST147 and co-producing NDM-1 and CTX-M-15, with a few isolates also harbouring blaOXA-48. Two sporadic isolates of K. pneumoniae ST307 and E. coli ST167 producing NDM-7 were also identified. blaNDM-1 was carried in two related IncR plasmid populations and blaNDM-7 in a conjugative 50 kb IncX3 plasmid. Conclusions We report the inter-hospital dissemination of XDR high-risk clones of K. pneumoniae and E. coli associated with the carriage of small, transferable plasmids harbouring blaNDM genes.

We determined the prevalence of skin mites in the hair follicles of the abdomen and caudal areas of 94 wild house mice (Mus musculus domesticus) from seven Canary Islands (Spain). We detected and i...

The identification of milk microbial communities in ruminants is relevant for understanding the association between milk microbiota and health status. The most common approach for studying the microbiota is amplifying and sequencing specific hypervariable regions of the 16S rRNA gene using massive sequencing techniques. However, the taxonomic resolution is limited to family and, in some cases, genus level. We aimed to improve taxonomic classification of the water buffalo milk microbiota by amplifying and sequencing the full-length 16S rRNA gene (1,500 bp) using Nanopore sequencing (single-molecule sequencing). When comparing with short-read results, we improved the taxonomic classification, reaching species level. We identified the main microbial agents of subclinical mastitis at the species level that were in accordance with the microbiological culture results. These results confirm the potential of single-molecule sequencing for in-depth analysis of microbial populations in dairy animals.

We have de novo assembled 67 Staphylococcus pseudintermedius genomes, with median values of 2.6 Mbp size and 99.43% completeness, 2,386 coding sequences, 19 complete rRNAs, 59 tRNAs, and 4 noncoding RNAs. We released 51 single-contig complete genomes and 16 genomes with a circular main contig using Nanopore sequencing.

The human gut microbiome has been extensively studied, yet the canine gut microbiome is still largely unknown. The availability of high-quality genomes is essential in the fields of veterinary medicine and nutrition to unravel the biological role of key microbial members in the canine gut environment. Our aim was to evaluate nanopore long-read metagenomics and Hi-C (high-throughput chromosome conformation capture) proximity ligation to provide high-quality metagenome-assembled genomes (HQ MAGs) of the canine gut environment. By combining nanopore long-read metagenomics and Hi-C proximity ligation, we retrieved 27 HQ MAGs and 7 medium-quality MAGs of a faecal sample of a healthy dog. Canine MAGs (CanMAGs) improved genome contiguity of representatives from the animal and human MAG catalogues – short-read MAGs from public datasets – for the species they represented: they were more contiguous with complete ribosomal operons and at least 18 canonical tRNAs. Both canine-specific bacterial species and gut generalists inhabit the dog’s gastrointestinal environment. Most of them belonged to Firmicutes , followed by Bacteroidota and Proteobacteria . We also assembled one Actinobacteriota and one Fusobacteriota MAG. CanMAGs harboured antimicrobial-resistance genes (ARGs) and prophages and were linked to plasmids. ARGs conferring resistance to tetracycline were most predominant within CanMAGs, followed by lincosamide and macrolide ones. At the functional level, carbohydrate transport and metabolism was the most variable within the CanMAGs, and mobilome function was abundant in some MAGs. Specifically, we assigned the mobilome functions and the associated mobile genetic elements to the bacterial host. The CanMAGs harboured 50 bacteriophages, providing novel bacterial-host information for eight viral clusters, and Hi-C proximity ligation data linked the six potential plasmids to their bacterial host. Long-read metagenomics and Hi-C proximity ligation are likely to become a comprehensive approach to HQ MAG discovery and assignment of extra-chromosomal elements to their bacterial host. This will provide essential information for studying the canine gut microbiome in veterinary medicine and animal nutrition.

Staphylococcus pseudintermedius is the main aetiological agent of canine pyoderma. Whole genome sequencing is the most comprehensive way of obtaining relevant genomic information about micro-organisms.Oxford Nanopore technology enables quality sequencing and de novo assembly of the whole genome of S. pseudintermedius. Whole genome analysis of S. pseudintermedius may help to better understand the pathogenesis of canine pyodermas.Twenty-two strains of S. pseudintermedius isolated from the skin of five healthy dogs and 33 strains isolated from skin of 33 dogs with pyoderma were analysed. DNA was extracted and sequenced using Oxford Nanopore MinION, a new technology that delivers longer reads in a hand-held device. The pangenome was analysed and visualised with Anvi'o 6.1.Nanopore technology allowed the sequencing and de novo assembly of the genomes of 55 S. pseudintermedius strains isolated from healthy dogs and from dogs with pyoderma. The average genome size of S. pseudintermedius was 2.62 Mbp, with 48% being core genome. Pyoderma isolates contained a higher number of antimicrobial resistance genes, yet the total number of virulence factors genes did not change between isolates from healthy dogs and from dogs with pyoderma. Genomes of meticillin-resistant S. pseudintermedius (MRSP) strains were larger than those of meticillin-susceptible (MSSP) strains (2.80 Mbp versus 2.59 Mbp), as a consequence of a greater presence of antimicrobial resistance genes, phages and prophages.This technique allows much more precise and easier characterisation of canine S. pseudintermedius populations and may lead to a better understanding of the pathogenesis of canine pyodermas.Staphylococcus pseudintermedius est le principal agent étiologique de la pyodermite canine. Le séquençage du génome entier est le moyen le plus complet d'obtenir des informations génomiques pertinentes sur les micro-organismes. HYPOTHÈSE/OBJECTIFS: La technologie Oxford Nanopore permet un séquençage de qualité et un assemblage de novo de l'ensemble du génome de S. pseudintermedius. L'analyse du génome entier de S. pseudintermedius peut aider à mieux comprendre la pathogénie des pyodermites canines. MÉTHODES ET MATERIEL: Vingt-deux souches de S. pseudintermedius isolées de la peau de cinq chiens sains et 33 souches isolées de la peau de 33 chiens atteints de pyodermite ont été analysées. L'ADN a été extrait et séquencé à l'aide d'Oxford Nanopore MinION, une nouvelle technologie qui offre des lectures plus longues dans un appareil portatif. Le pangénome a été analysé et visualisé avec ANVI'O 6.1. RÉSULTATS: La technologie Nanopore a permis le séquençage et l'assemblage de novo des génomes de 55 souches de S. pseudintermedius isolées de chiens sains et de chiens atteints de pyodermite. La taille moyenne du génome de S. pseudintermedius était de 2,62 Mpb, dont 48 % étaient le génome central. Les isolats de pyodermite contenaient un nombre plus élevé de gènes de résistance aux antimicrobiens, mais le nombre total de gènes de facteurs de virulence n'a pas changé entre les isolats de chiens sains et de chiens atteints de pyodermite. Les génomes des souches de S. pseudintermedius résistantes à la méticilline (MRSP) étaient plus grands que ceux des souches sensibles à la méticilline (MSSP) (2,80 Mbp contre 2,59 Mbp), en raison d'une plus grande présence de gènes de résistance aux antimicrobiens, de phages et de prophages.Cette technique permet une caractérisation beaucoup plus précise et plus facile des populations canines de S. pseudintermedius et peut conduire à une meilleure compréhension de la pathogénie des pyodermites canines.INTRODUCCIÓN: Staphylococcus pseudintermedius es el principal agente etiológico de la pioderma canina. La secuenciación del genoma completo es la forma más completa de obtener información genómica relevante sobre microorganismos. HIPÓTESIS/OBJETIVOS: la tecnología Oxford Nanopore permite la secuenciación de calidad y el ensamblaje de novo de todo el genoma de S. pseudintermedius. El análisis del genoma completo de S. pseudintermedius puede ayudar a comprender mejor la patogenia de las piodermas caninas. MÉTODOS Y MATERIALES: Se analizaron veintidós cepas de S. pseudintermedius aisladas de la piel de cinco perros sanos y 33 cepas aisladas de la piel de 33 perros con pioderma. El DNA se extrajo y secuenció utilizando Oxford Nanopore MinION, una nueva tecnología que ofrece lecturas más largas en un dispositivo de mano. El pangenoma se analizó y visualizó con ANVI’O 6.1. RESULTADOS: la tecnología Nanopore permitió la secuenciación y el ensamblaje de novo de los genomas de 55 cepas de S. pseudintermedius aisladas de perros sanos y de perros con pioderma. El tamaño medio del genoma de S. pseudintermedius fue de 2,62 Mbp, con un 48% de genoma central. Los aislados de pioderma contenían un mayor número de genes de resistencia a los antimicrobianos, sin embargo, el número total de genes de factores de virulencia no cambió entre los aislados de perros sanos y los de perros con pioderma. Los genomas de las cepas de S. pseudintermedius resistentes a la meticilina (MRSP) fueron más grandes que los de las cepas sensibles a la meticilina (MSSP) (2,80 Mbp versus 2,59 Mbp), como consecuencia de una mayor presencia de genes de resistencia a los antimicrobianos, fagos y profagos. CONCLUSIONES E IMPORTANCIA CLÍNICA: esta técnica permite una caracterización mucho más precisa y sencilla de las poblaciones caninas de S. pseudintermedius y puede conducir a una mejor comprensión de la patogenia de las piodermas caninas.Staphylococcus pseudintermedius stellt die hauptsächliche ätiologische Ursache für die canine Pyodermie dar. Eine Gesamtgenomsequenzierung ist der umfassendste Weg, um relevante Genominformation über Mikroorganismen zu erhalten.Die Oxford Nanopore Technologie ermöglicht eine qualitative Sequenzierung und eine de novo Anordnung des Gesamtgenoms von S. pseudintermedius. Die Gesamtgenomsequenzierung von S. pseudintermedius könnte dabei helfen, die Pathogenese der caninen Pyodermie besser zu verstehen.Zweiundzwanzig Stämme von S. pseudintermedius, die von der Haut von fünf gesunden Hunden und 33 Stämme, die von 33 Hunden mit Pyodermie isoliert worden waren, wurden analysiert. DNA wurde extrahiert und mittels Oxford Nanopore MinION, einer neuen Technologie, die längere Sequenzen in einem Handgerät liefert, sequenziert. Das Pangenom wurde analysiert und mittels ANVI´O 6.1. analysiert.Die Nanopore Technologie ermöglichte eine Sequenzierung und de novo Anordnung der Genome von 55 S. pseudintermedius Stämmen, die von gesunden Hunden und von Hunden mit Pyodermie isoliert worden waren. Die durchschnittliche Genom Länge von S. pseudintermedius betrug 2,62 Mbp, wobei 48% davon das Hauptgenom ausmachte. Pyodermie Isolate beinhalteten eine größere Anzahl an antimikrobiellen Resistenzgenen; die Gesamtzahl der Virulenzfaktorgene unterschied sich allerdings nicht zwischen den Isolaten von gesunden Hunden und von Hunden mit Pyodermie. Genome von Methicillin-resistenten S. pseudintermedius (MRSP) Stämmen waren größer als jene der Methicillin-sensiblen (MSSP) Stämme (2,8 Mbp versus 2,59 Mbp), was eine Folge der vermehrten Präsenz von antimikrobiellen Resistenzgenen, Phagen und Prophagen war.Diese Technik erlaubt eine wesentlich präzisere und einfachere Charakterisierung der caninen S. pseudintermedius Populationen und könnte zu einem besseren Verstehen der Pathogenese der caninen Pyodermien führen.背景: Staphylococcus pseudintermediusは、犬の膿皮症の主な病因です。全ゲノムシーケンスは、微生物に関する関連するゲノム情報を取得するための最も包括的な方法です。 仮説/目的: Oxford Nanoporeテクノロジーにより、S。pseudintermediusの全ゲノムの高品質なシーケンスとdenovoアセンブリが可能になります。 S. pseudintermediusの全ゲノム解析は、犬の膿皮症の病因をよりよく理解するのに役立つ可能性があります。 方法と材料: 5匹の健康な犬の皮膚から分離されたS.pseudintermediusの22株と、膿皮症の33匹の犬の皮膚から分離された33株が分析されました。 DNAは、ハンドヘルドデバイスでより長い読み取りを実現する新しいテクノロジーであるOxford Nanopore MinIONを使用して抽出およびシーケンスされました。パンゲノムはANVI’O6.1で分析および視覚化されました。 結果: Nanoporeテクノロジーにより、健康な犬と膿皮症の犬から分離された55のS.pseudintermedius株のゲノムの配列決定とdenovoアセンブリが可能になりました。 S.pseudintermediusの平均ゲノムサイズは2.62Mbpで、48％がコアゲノムでした。膿皮症分離株は、より多くの抗菌薬耐性遺伝子を含んでいましたが、病原性因子遺伝子の総数は、健康な犬と膿皮症の犬からの分離株間で変化しませんでした。メチシリン耐性S.pseudintermedius（MRSP）株のゲノムは、抗菌剤耐性遺伝子、ファージ、およびプロファージの存在が大きい結果として、メチシリン感受性（MSSP）株のゲノムよりも大きかった（2.80Mbp対2.59Mbp）。 結論と臨床的重要性: この手法により、犬のS. pseudintermedius集団のより正確で簡単な特性評価が可能になり、犬の膿皮症の病因のより良い理解につながる可能性があります。.背景: 假中间型葡萄球菌是犬脓皮病的主要病原。全基因组测序是获得微生物相关基因组信息最全面的方式。 假设/目的: 纳米孔技术能够对假中间型葡萄球菌的全基因组进行质量测序和从头组装。对假中间型葡萄球菌进行全基因组分析可能有助于更好地了解犬脓皮病的发病机制。 方法和材料: 从5只健康犬皮肤中分离的22株，以及从33只脓皮病犬皮肤中分离的33株假中间型葡萄球菌，对其进行分析。使用纳米孔 MinION提取DNA并测序，这是一种手持设备，这种新技术可传递更长的测序片段。用ANVI'O 6.1分析全基因组并将其可视化。 结果: 从健康犬和脓皮病犬中分离的55株假中间型葡萄球菌，用纳米孔技术对其基因组进行测序和从头组装。假中间型葡萄球菌的平均基因组大小为2.62 Mbp，其中48%为核心基因组。脓皮病分离株含有较多数量的抗菌药物耐药基因，然而毒力因子基因的总数在健康犬和脓皮病犬的分离株之间没有变化。耐甲氧西林假中间型葡萄球菌(MRSP)菌株的基因组比甲氧西林敏感(MSSP)菌株的基因组大（2.80 Mbp与2.59 Mbp），这是由于存在更多抗菌药物耐药基因、噬菌体和前噬菌体。 结论和临床重要性: 该技术可更精确和更容易地表征犬假中间型葡萄球菌种群，并可能有助于更好地了解犬脓皮病的发病机制。.Staphylococcus pseudintermedius é o principal agente etiológico da piodermite canina. O sequenciamento completo do genoma é a forma mais aprofundada de se obter informações genômicas relevantes sobre microrganismos. HIPÓTESE/OBJETIVOS: A tecnologia de Oxford Nanopore permite o sequenciamento de boa qualidade e montagem “de novo” do genoma completo de S. pseudintermedius. A análise do genoma completo de S. pseudintermedius pode auxiliar na melhor compreensão da patogênese da piodermite canina. MÉTODOS E MATERIAIS: Vinte e duas cepas de S. pseudintermedius isoladas da pele de cinco cães saudáveis e 33 cepas isoladas da pele de 33 cães com piodermite foram analisadas. O DNA foi extraído e sequenciado utilizando Oxford Nanopore MinION, uma tecnologia nova que fornece leituras mais longas em um aparelho portátil. O pangenoma foi analisado e visualizado com ANVI’O 6.1.A tecnologia Nanopore permitiu o sequenciamento e a montagem “de novo” dos genomas de 55 cepas de S. pseudintermedius isoladas de cães saudáveis de cães com piodermite. A média do tamanho do genoma de S. pseudintermedius foi de 2,62 Mbp, sendo 48% de genoma base. Os isolados de piodermite continham um maior número de genes de resistência antimicrobiana, apesar de o número total de fatores de virulência não sofreu alteração entre os isolados de cães saudáveis e de cães com piodermite. Os genomas das cepas S. pseudintermedius resistentes à meticilina (MRSP) foram maiores que aqueles das cepas suscetíveis à meticilina (MSSP) (2,80 Mbp versus 2,59 Mbp), como consequência da maior presença de genes de resistência antimicrobiana, fagos e profagos. CONCLUSÕES E IMPORTÂNCIA CLÍNICA: A técnica testada permite uma caracterização muito mais simples e precisa das populações de S. pseudintermedius canino e pode levar ao melhor entendimento da patogênese da piodermite canina.

Long-read metagenomics facilitates the assembly of high-quality metagenome-assembled genomes (HQ MAGs) out of complex microbiomes. It provides highly contiguous assemblies by spanning repetitive regions, complete ribosomal genes, and mobile genetic elements. Hi-C proximity ligation data bins the long contigs and their associated extra-chromosomal elements to their bacterial host. Here, we characterized a canine fecal sample combining a long-read metagenomics assembly with Hi-C data, and further correcting frameshift errors.We retrieved 27 HQ MAGs and seven medium-quality (MQ) MAGs considering MIMAG criteria. All the long-read canine MAGs improved previous short-read MAGs from public datasets regarding contiguity of the assembly, presence, and completeness of the ribosomal operons, and presence of canonical tRNAs. This trend was also observed when comparing to representative genomes from a pure culture (short-read assemblies). Moreover, Hi-C data linked six potential plasmids to their bacterial hosts. Finally, we identified 51 bacteriophages integrated into their bacterial host, providing novel host information for eight viral clusters that included Gut Phage Database viral genomes. Even though three viral clusters were species-specific, most of them presented a broader host range.In conclusion, long-read metagenomics retrieved long contigs harboring complete assembled ribosomal operons, prophages, and other mobile genetic elements. Hi-C binned together the long contigs into HQ and MQ MAGs, some of them representing closely related species. Long-read metagenomics and Hi-C proximity ligation are likely to become a comprehensive approach to HQ MAGs discovery and assignment of extra-chromosomal elements to their bacterial host.

We have de novo assembled the genome sequence of Malassezia pachydermatis isolated from a canine otitis sample with Nanopore-only long reads. With 99× coverage and 8.23 Mbp, the genome sequence was assembled in 10 contigs, with 6 of them corresponding to chromosomes, improving the scaffolding of previous genome assemblies for the species.

We have de novo assembled and polished 61 Staphylococcus pseudintermedius genome sequences with Nanopore-only long reads. Completeness was 99.25%. The average genome size was 2.70 Mbp, comprising 2,506 coding sequences, 19 complete rRNAs, 56 to 59 tRNAs, and 4 noncoding RNAs (ncRNAs), as well as CRISPR arrays. Fil: Francino, Olga. Universitat Autònoma de Barcelona; España Fil: Pérez, Daniel. Universitat Autònoma de Barcelona; España Fil: Viñes, Joaquim. Universitat Autònoma de Barcelona; España Fil: Fonticoba, Rocío. Universitat Autònoma de Barcelona; España Fil: Madroñero, Sergi. Universitat Autònoma de Barcelona; España Fil: Meroni, Gabrielle. No especifíca; Fil: Martino, P.. No especifíca; Fil: Martínez, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Cuscó, Anna. Universitat Autònoma de Barcelona; España Fil: Fàbregas, Norma. Universitat Autònoma de Barcelona; España Fil: Migura García, Lourdes. Universitat Autònoma de Barcelona; España Fil: Ferrer, Lluís. Universitat Autònoma de Barcelona; España

Colistin use has mostly been stopped in human medicine, due to its toxicity. However, nowadays, it still is used as a last-resort antibiotic to treat hospital infections caused by multi-drug resistant Enterobacteriaceae. On the contrary, colistin has been used in veterinary medicine until recently. In this study, 210 fecal samples from pigs (n = 57), calves (n = 152), and the farmer (n = 1) were collected from a farm where E. coli harboring mcr-1-mcr-3 was previously detected. Samples were plated, and mcr-genes presence was confirmed by multiplex-PCR. Hybrid sequencing which determined the presence and location of mcr-1, other antibiotic resistance genes, and virulence factors. Eighteen colistin resistant isolates (13 from calves, four from pigs, and one from the farmer) contained mcr-1 associated with plasmids (IncX4, IncI2, and IncHI2), except for two that yielded mcr-1 in the chromosome. Similar plasmids were distributed in different E. coli lineages. Transmission of mcr-1 to the farmer most likely occurred by horizontal gene transfer from E. coli of calf origin, since plasmids were highly similar (99% coverage, 99.97% identity). Moreover, 33 virulence factors, including stx2 for Shiga toxin E. coli (STEC) were detected, highlighting the role of livestock as a reservoir of pathotypes with zoonotic potential.

Canine hip dysplasia is one of the most prevalent developmental orthopedic diseases in dogs worldwide. Unfortunately, the success of eradication programs against this disease based on radiographic diagnosis is low. Adding the use of diagnostic genetic tools to the current phenotype-based approach might be beneficial. The aim of this study was to develop a genetic prognostic test for early diagnosis of hip dysplasia in Labrador Retrievers. To develop our DNA test, 775 Labrador Retrievers were recruited. For each dog, a blood sample and a ventrodorsal hip radiograph were taken. Dogs were divided into two groups according to their FCI hip score: control (A/B) and case (D/E). C dogs were not included in the sample. Genetic characterization combining a GWAS and a candidate gene strategy using SNPs allowed a case-control population association study. A mathematical model which included 7 SNPs was developed using logistic regression. The model showed a good accuracy (Area under the ROC curve = 0.85) and was validated in an independent population of 114 dogs. This prognostic genetic test represents a useful tool for choosing the most appropriate therapeutic approach once genetic predisposition to hip dysplasia is known. Therefore, it allows a more individualized management of the disease. It is also applicable during genetic selection processes, since breeders can benefit from the information given by this test as soon as a blood sample can be collected, and act accordingly. In the authors' opinion, a shift towards genomic screening might importantly contribute to reducing canine hip dysplasia in the future. In conclusion, based on genetic and radiographic information from Labrador Retrievers with hip dysplasia, we developed an accurate predictive genetic test for early diagnosis of hip dysplasia in Labrador Retrievers. However, further research is warranted in order to evaluate the validity of this genetic test in other dog breeds.

Background. Metagenomics is a powerful and rapidly developing approach that provides new biological insights into the microbes inhabiting underexplored environments, such as canine fecal microbiome. We investigate long-read metagenomics with Nanopore sequencing to profile the fecal microbiome and to retrieve high-quality metagenome-assembled genomes (HQ MAGs) from a healthy dog.Results. More than 99% of total classified reads corresponded to Bacteria. The most abundant phylum was Bacteroidetes (~80% of total reads), followed by Firmicutes, Proteobacteria, and Fusobacteria. Prevotella (>50%) and Bacteroides (>20%) are the more abundant genera, followed by Fusobacterium, Megamonas, Sutterella, and other fecal-related genera, (each representing Succinivibrio, Sutterella, Prevotellamassilia, Phascolarctobacterium, Enterococcus, Blautia, and Catenibacterium genera. Succinivibrio HQ MAG represents a novel candidate bacterial species. Sutterella HQ MAG is potentially the first reported genome assembly for Sutterella stercoricanis, as assigned by 16S rRNA gene similarity. Prevotellamassilia, Phascolarctobacterium, Catenibacterium, and Blautia sp900541345 HQ MAGs improved the contiguity of previously reported genome assemblies in their respective genera, and the number of rRNA genes and tRNA genes. Finally, Enterococcus hirae and Blautia sp003287895 HQ MAGs represented species that already have a complete reference genome. At the technical level, we demonstrated that a high-molecular weight DNA extraction improved the taxonomic classification of the raw unassembled reads, the metagenomics assembly contiguity, and the retrieval of longer and circular contigs, which are potential HQ MAGs. Conclusions. Long-read metagenomics allowed us to recover HQ MAGs from canine feces of a healthy dog. The high-molecular weight DNA extraction to improve contiguity and the correction of the insertions and deletions to reduce frameshift errors ensure the retrieval of complete single-contig HQ MAGs.

Background. Studies using highly sensitive molecular techniques have detected bacterial communities below the human epidermis. Depending on their abundance and composition, this finding could be clinically relevant. This possibility, however, has not been investigated in the dog so far. The aim of this study was to determine if bacteria can be detected in the dermis and subcutaneous tissue of healthy dogs using two different approaches: traditional cultures and massive DNA sequencing of the V4 region of bacterial 16S rRNA gene using next-generation sequencing (NGS).Results. Seven healthy dogs were included in the study, and two sets of samples were collected from each subject. Sample sets were composed of a 6-mm abdominal skin biopsy, including epidermis, dermis, and subcutis, a skin surface swab, and an environmental blank sample for contamination control. One set of samples from each dog was submitted for bacterial culture and the other one for bacterial DNA amplification and sequencing. Five different bacterial genera (Staphylococcus, Bacillus, Corynebacterium, Streptococcus, and Enterococcus) were isolated in five out of the seven skin surface swab samples with traditional culture methods, while no growth was obtained from the other two samples. Although some DNA could be amplified from epidermal, dermal, and subcutaneous tissue samples, the results of the NGS were similar to those of the blanks.Conclusion. When investigated with traditional culture methods, the dermis and subcutaneous tissue of healthy dogs are sterile. NGS techniques lead to the detection of some bacterial DNA which, although similar to the signal detected in blanks, does not support the presence of a microbiome in dermis or subcutaneous tissue.

Studies on tick microbial communities historically focused on tick-borne pathogens. However, there is an increasing interest in capturing relationships among non-pathogenic endosymbionts and exploring their relevance for tick biology. The present study included a total of 1600 adult ticks collected from domestic dogs in 4 different biogeographical regions of Spain. Each pool formed by 1 to 10 halves of individuals representing one specific ticks species was examined by PCR for the presence of Coxiellaceae, Rickettsia spp., Rickettsiales, Wolbachia spp., and other bacterial DNA. Of the pools analyzed, 92% tested positive for endosymbiont-derived DNA. Coxiella spp. endosymbionts were the most prevalent microorganisms, being always present in Rhipicephalus sanguineus sensu lato (s.l.) pools. Rickettsia spp. DNA was detected in 60% of Dermacentor reticulatus pools and 40% of R. sanguineus s.l. pools, with a higher diversity of Rickettsia species in R. sanguineus s.l. pools. Our study reveals a negative relationship of Rickettsia massiliae with the presence of tick-borne pathogens in the same pool of ticks. An additional endosymbiont, 'Candidatus Rickettsiella isopodorum', was only detected in D. reticulatus pools. Data from this study indicate that dogs in Spain are exposed to several endosymbionts. Due to the importance of tick-borne pathogens, characterizing the role of endosymbionts for tick physiology and prevalence, may lead to novel control strategies.

The availability of long-read technologies, like Oxford Nanopore Technologies, provides the opportunity to sequence longer fragments of the fungal ribosomal operon, up to 6 Kb (18S-ITS1-5.8S-ITS2-28S), and to improve the taxonomy assignment of the communities up to the species level and in real-time. We assess the taxonomy skills of amplicons targeting a 3.5 Kb region (V3 18S-ITS1-5.8S-ITS2-28S D2) and a 6 Kb region (V1 18S-ITS1-5.8S-ITS2-28S D12) with the What’s in my pot (WIMP) classifier. We used the ZymoBIOMICS™ mock community and different microbiological fungal cultures as positive controls. Long amplicon sequencing correctly identified Saccharomyces cerevisiae and Cryptococcus neoformans from the mock community and Malassezia pachydermatis, Microsporum canis, and Aspergillus fumigatus from the microbiological cultures. Besides, we identified Rhodotorula graminis in a culture mislabeled as Candida spp.We applied the same approach to external otitis in dogs. Malassezia was the dominant fungal genus in dogs’ ear skin, whereas M. pachydermatis was the main species in the healthy sample. Conversely, we identified a higher representation of M. globosa and M. sympodialis in otitis affected samples. We demonstrate the suitability of long ribosomal amplicons to characterize the fungal community of complex samples, either healthy or with clinical signs of infection.

Objectives To investigate evidence for selected vector-borne pathogen infections in dogs with pericardial effusion living in a Mediterranean area in which several canine vector-borne diseases are endemic. Materials and methods Archived EDTA blood (n=68) and pericardial fluid samples (n=58) from dogs with pericardial effusion (n=68) were included. Dogs without pericardial effusion examined for other reasons were included as controls (n=60). Pericardial effusion was classified as neoplastic in 40 dogs, idiopathic in 23 dogs and of unknown aetiology in 5 dogs. Real-time PCR was performed for Leishmania infantum, Ehrlichia/Anaplasma species, Hepatozoon canis, Babesia species, Rickettsia species and Bartonella species, and sequencing of PCR products from positive samples was used to confirm species specificity. Results Vector-borne pathogens were found in 18 dogs: 16 of 68 dogs with pericardial effusion (23·5%) and two of 60 control dogs (3·3%). Positive dogs demonstrated DNA of Leishmania infantum (n=7), Anaplasma platys (n=2, one dog coinfected with Leishmania infantum), Babesia canis (n=5), Babesia gibsoni (n=3) and Hepatozoon canis (n=2). Vector-borne pathogens were more commonly detected among dogs with pericardial effusion than controls (P=0·001). There was no relationship between aetiology of the pericardial effusion and evidence of vector-borne pathogens (P=0·932). Clinical significance Vector-borne pathogens are often detected in dogs with pericardial effusion and require further investigation, especially in dogs with idiopathic pericardial effusion. PCR can provide additional information about the potential role of vector-borne pathogens in dogs with pericardial effusion living in endemic areas.

Here we report the genome assembly, using a hybrid approach with Illumina and Nanopore sequencing, of a pathogenic Staphylococcus pseudintermedius strain isolated from a case of canine otitis. Genome assembly confirmed the antimicrobial resistance profile (disk diffusion testing) with specific genes and mutations.

The availability of long-read technologies, like Oxford Nanopore Technologies, provides the opportunity to sequence longer fragments of the fungal ribosomal operon, up to 6 Kb (18S-ITS1-5.8S-ITS2-28S) and to improve the taxonomy assignment of the communities up to species level and in real-time. We assess the applicability for taxonomic assignment of amplicons targeting a 3.5 Kb region (V3 18S-ITS1-5.8S-ITS2-28S D2) and a 6 Kb region (V1 18S-ITS1-5.8S-ITS2-28S D12) with the What’s in my pot (WIMP) classifier. We used the ZymoBIOMICSTM mock community and different microbiological fungal cultures as positive controls. Long amplicon sequencing correctly identified Saccharomyces cerevisiae and Cryptococcus neoformans from the mock community and Malassezia pachydermatis, Microsporum canis and Aspergillus fumigatus from the microbiological cultures. Besides, we identified Rhodotorula graminis in a culture mislabelled as Candida spp. We applied the same approach to external otitis in dogs. Malassezia was the dominant fungal genus in dogs’ ear skin, whereas Ma. pachydermatis was the main species in the healthy sample. Conversely, we identified a higher representation of Ma. globosa and Ma. sympodialis in otitis affected samples. We demonstrate the suitability of long ribosomal amplicons to characterize the fungal community of complex samples, either healthy or with clinical signs of infection.

One-Health studies applying massive-parallel and single-molecule sequencing are a suitable approximation to try to understand how antibiotic resistances flow between the human-animal-environment scenario. Colistin has been withdrawn in human medicine due to its toxicity, limiting its usage as a last-resort treatment option for multidrug-resistant Gram-negative bacteria. However, it is still used orally to treatEnterobacteriaceaeinfections in veterinary medicine. Since 2015, colistin resistance appeared to be located in mobile genetic elements, raising the concern of the likelihood of transmission by horizontal gene transfer between animals and humans. In this study, 202 faecal samples were collected in a mixed farm from pigs, calves, and the farmer. PCR forthe mcr-1 gene was positive for 18 of the isolates, and Nanopore sequencing allowed us to determine the location of the gene, either on the chromosome or in plasmids. Three types of replicons were found within the positive isolates harbouring themcr-1: IncX4, IncI2, and IncHI2. Four different genetic contexts probably indicate different stages of gene stabilization, either in the chromosome or plasmid, withISApl1as the main insertion element flanking the gene. Moreover, 43 other resistance genes were found in our samples, related to more than six different antibiotic families (e.g. aminoglycosides, lincosamides, beta-lactams, macrolides, trimethoprim, phenicols, and sulphonamides). We found resistance genes against colistin and that six antibiotic families together in at least one of the isolates from human, swine, and bovine. Isolate 15B-22 harboured one plasmid with seven resistance genes related to four families of antibiotics other than polymyxins, meaning that there are more chances to maintain colistin resistance even with the withdrawn of colistin. Nanopore long reads allowed us to assemble the DNA elements within the isolates easily and determine the genetic context of themcr-1gene. Furthermore, they allowed us to describe and locate more antimicrobial resistance genes to other antibiotic families and antiseptic compounds.

Long-read metagenomics –using single-molecule sequencers– has the potential to assembly entire genomes, even from complex metagenomics datasets. Using long-read metagenomics with Nanopore sequencing in pooled samples, we aim to improve the individual taxonomic profiles obtained with V4 16S rRNA massive sequencing and to assemble the fecal metagenome of healthy dogs.Fecal samples from healthy dogs were sequenced individually using V4 16S rRNA gene and in pools using a shallow metagenomics approach with Nanopore sequencing. Long-read metagenomics allowed us refining the V4 16S rRNA results up to the species level and determining the main bacterial species inhabiting on fecal microbiota of our cohort of healthy dogs. Among them, the most abundant were Fusobacterium varium; Megamonas hypermegale; Bacteroides vulgatus; Blautia hansenii; Clostridium perfringens; and Clostridoides difficile. Once performed the metagenome assembly, one contig was suggested to be circular and hit to an uncultured crAssphage.To conclude, shallow long-read metagenomics with pooled samples using MinION allowed characterizing the dog fecal microbiota at lower taxonomic level, such as bacterial species. The assembly of the reads retrieved a contig that represents a circular draft genome of an uncultured CrAssphage from dog fecal samples that is one of the most abundant bacteriophages in the human gut.

Canine otitis externa (OE) is a common disease characterised by inflammation of the epithelial tissue of the external ear canal. Secondary infections are frequent, and Malassezia pachydermatis and Staphylococcus pseudintermedius are routinely isolated and treated with antifungal and antibiotic compounds.To analyse the otitis ear microbiome before and after a treatment with prednisolone plus pomegranate or antimicrobial drugs ANIMALS: 15 dogs with nonpurulent OE.A 30 day, double-blinded, multicentre, randomized and controlled parallel-group (1:1) trial was conducted in 15 dogs with nonpurulent OE, following two different topical treatments (prednisolone plus pomegranate versus prednisolone plus antibiotic and antifungal drugs). On days (D)0, D15 and D30, serum and skin otic samples were collected, and clinical examination and microbiome analysis (bacteria and fungi) were performed. Results were compared with validated otitis clinical scores to assess the effectiveness of both treatments.Nine bacterial and four fungal families were detected during the three time-points tested. An increase in fungal diversity (Shannon index) and composition was the most significant change observed after both treatments. At treatment D15 and D30, the reduction in clinical signs was statistically significant in both treatment groups (P ≤ 0.05). Prednisolone plus pomegranate cleanser treatment was able to control the clinical signs of otitis as well as the bacterial and fungal overgrowth.Mild otitis cases associated with microbial overgrowth may be managed with topical antiseptic and anti-inflammatory agents without the need for antibiotic and/or antifungal compounds.L’otite externe canine (OE) est une maladie fréquente caractérisée par une inflammation du tissu épithélial du conduit auriculaire externe. Les infections secondaires sont fréquentes et Malassezia pachydermatis et Staphylococcus pseudintermedius sont isolés en routine et traités par des antibiotiques et antifongiques. HYPOTHÈSES/OBJECTIFS: Analyser le microbiome auriculaire avant et après traitement avec prednisolone et pomegranate ou antimicrobiens.15 chiens avec OE non-purulente. MATÉRIELS ET METHODS: Un essai de 30 jours, en double aveugle, multicentrique, randomisé et contrôlé en parallèle (1 :1) a été réalisé chez 15 chiens avec OE non purulente, a l’aide de deux traitements topiques différents (prednisolone plus pomegranate versus prednisolone plus antibiotique et antifongique). A jours (J)0, J15 et J30, les échantillons de serum et de peau auriculaire ont été prélevés et un examen clinique et une analyse du microbiome (bactérie et champignon) ont été réalisés. Les résultats ont été comparés avec les scores validés d’otite pour déterminer l’efficacité des deux traitements. RÉSULTATS: Neuf bactéries et quatre champignons ont été détectés au cours des trois points testés. Une augmentation de la diversité fongique (Shannon index) et de la composition a été le changement le plus significatif observé après les deux traitements. Aux traitements à J15 et J30, la réduction des signes cliniques était statistiquement significative dans les deux groupes de traitement (P ≤ 0.05). Le traitement avec prednisolone plus pomegranate a été capable de contrôler les signes cliniques d’otite aussi bien que la surpopulation bactérienne et fongique.Les cas modérés d’otite associés à une surpopulation microbienne peuvent être gérés avec des antiseptiques topiques et des anti-inflammatoires sans besoin d’antibiotique et/ou d’antifongiques.INTRODUCCIÓN: la otitis externa canina (OE) es una enfermedad común caracterizada por la inflamación del tejido epitelial del conducto auditivo externo. Las infecciones secundarias son frecuentes, y Malassezia pachydermatis y Staphylococcus pseudintermedius se aíslan de forma rutinaria y se tratan con compuestos antifúngicos y antibióticos. HIPÓTESIS/OBJETIVOS: Analizar el microbioma del oído en casos de otitis antes y después de un tratamiento con prednisolona más extracto de granada o más fármacos antimicrobianos ANIMALES: 15 perros con OE no purulenta. MÉTODOS Y MATERIALES: se realizó un ensayo de 30 días, doble ciego, multicéntrico, al azar y controlado de grupos paralelos (1:1) en 15 perros con OE no purulenta después de dos tratamientos tópicos diferentes (prednisolona más extracto de granada versus prednisolona más fármacos antibióticos y antimicóticos). En los días (D) 0, D15 y D30, se recogieron las muestras de suero y piel ótica, y se realizaron examen clínico y análisis del microbioma (bacterias y hongos). Los resultados se compararon con puntuaciones clínicas validadas por otitis para evaluar la eficacia de ambos tratamientos. RESULTADOS: Se detectaron nueve familias de bacterias y hongos en los tres tiempos evaluados. Un aumento en la diversidad fúngica (índice de Shannon) y de la composición fue el cambio más significativo observado después de ambos tratamientos. En los días de tratamiento D15 y D30, la reducción de los signos clínicos fue estadísticamente significativa en ambos grupos de tratamiento (P≤0,05). El tratamiento con prednisolona más un limpiador con extracto de granada pudo controlar los signos clínicos de la otitis, así como el crecimiento excesivo de bacterias y hongos. CONCLUSIONES E IMPORTANCIA CLÍNICA: los casos de otitis leve asociados con el sobrecrecimiento microbiano pueden tratarse con agentes antisépticos y antiinflamatorios tópicos sin necesidad de antibióticos y/o compuestos antifúngicos.Die Otitis externa (OE) des Hundes ist eine häufige Erkrankung, die durch eine Entzündung des epithelialen Gewebes des äußeren Ohrkanals charakterisiert wird. Sekundäre Infektionen kommen dabei häufig vor, wobei Malassezia pachydermatis und Staphylococcus pseudintermedius routinemäßig isoliert und mit Antimykotika und Antibiotika behandelt werden.Eine Analyse des Mikrobioms des Otitis Ohrs vor und nach der Behandlung mit Prednisolon und Granatapfel oder antimikrobiellen Wirkstoffen.15 Hunde mit nicht-purulenter OE.Eine 30 tägige, doppel-blinde, multizenter, randomisierte Studie mit einer kontrollierten Parallel-Gruppe (1:1) wurde an 15 Hunden mit einer nicht-purulenten OE durchgeführt, wobei zwei verschiedene topische Behandlungen (Prednisolon und Granatapfel versus Prednisolon und Antibiotikum und Antimykotikum) verabreicht wurden. An den Tagen (D) 0, D15 und D30 wurden Serumproben und Proben aus dem Ohrkanal entnommen und eine klinische Untersuchung und Mikrobiom Analyse (Bakterien und Pilze) durchgeführt. Die Ergebnisse wurden mit validierten klinischen Otitis Werten verglichen, um die Wirksamkeit beider Behandlungen zu beurteilen.Neun Bakterienstämme und vier Pilzfamilien wurden zu den drei getesteten Zeitpunkten gefunden. Eine Zunahme der Pilz Vielfalt (Shannon Index) und ihrer Zusammensetzung war der signifikanteste Unterschied, der zwischen den beiden Behandlungen beobachtet werden konnte. Bei der Behandlung am D15 und D30 war die Reduzierung der klinischen Zeichen in beiden Gruppen statistisch signifikant (P≤ 0,05). Prednisolon und Granatapfelreiniger konnte die klinischen Zeichen der Otitis genauso gut kontrollieren wie das Überwachsen mit Bakterien und Pilzen.Fälle mit einer milden Otitis im Zusammenhang mit mikrobiellem Überwachstum könnten mit einem topischen Antiseptikum und entzündungshemmenden Wirkstoffen, ohne Antibiotika und/oder Antimykotika zu benötigen, gemanagt werden.背景-犬の外耳炎（OE）は、外耳道の上皮組織の炎症を特徴とする一般的な疾患である。二次感染は頻繁に起こり、Malassezia pachydermatiおよびStaphylococcus pseudintermediusは日常的に分離され、抗真菌性および抗菌化合物で治療される。 仮説/目的-本研究の目的は、プレドニゾロンおよびザクロ、または、プレドニゾロンおよび抗菌薬による治療前後の外耳炎を起こした耳のマイクロバイオームを解析することであった。 供試動物-非化膿性OE罹患犬15頭。 材料と方法- 30日間の二重盲検、多施設、ランダム化および対照並行群間（1：1）試験が、2つの異なる局所治療（プレドニゾロン+ザクロvsプレドニゾロン+抗生物質および抗真菌剤）に続いて、非化膿性OE罹患犬15頭で実施された。 （D）0、D15、およびD30日に、血清および耳の皮膚サンプルを収集し、臨床検査およびマイクロバイオーム解析（細菌および真菌）を実施した。結果を検証済みの中耳炎臨床スコアと比較して、両方の治療の有効性を評価した。 結果-テストした3つの時点で、9つの細菌ファミリーおよび4つの真菌ファミリーが検出された。真菌の多様性（シャノン指数）および組成の増加は、両方の治療後に観察された最も重要な変化であった。治療D15およびD30では、臨床徴候の減少は両方の治療群で統計的に有意であった（P≤0.05）。プレドニゾロンおよびザクロの洗浄治療は、耳炎の臨床兆候だけでなく、細菌や真菌の異常増殖を制御することができた。 結論と臨床的重要性-微生物の異常増殖に関連する軽度の耳炎症例は、抗生物質および/または抗真菌化合物を必要とせずに、局所消毒剤および抗炎症剤で管理できる。.背景-犬外耳炎(OE)是一种以外耳道上皮组织炎症为特征的常见疾病。继发感染频繁，常规分离出厚皮马拉色菌和假中间型葡萄球菌，并需要联合抗真菌药和抗生素治疗。 假设/目的-分析耳炎病例用泼尼松龙加石榴或抗菌药治疗前后的耳微生物组。 动物-15只非化脓性OE患犬。 方法和材料-对15只非化脓性OE患犬进行为期30天的双盲、多中心、随机和对照平行组(1:1)试验，采用两种不同的外部治疗（泼尼松龙加石榴与泼尼松龙加抗生素和抗真菌药）。在第(D)0、15和30天，采集血清和皮肤耳道样本，并进行临床检查和微生物组分析（细菌和真菌）。将结果与公认的耳炎临床评分进行比较，以评估两种治疗的有效性。 结果-在检测的3个时间点检测到9个细菌和4个真菌科。真菌多样性（Shannon指数）及其组成的增加是两种治疗后观察到的最显著变化。在给药D15和D30，两个给药组的临床症状降低均具有统计学显著性(P≤0.05)。泼尼松龙加石榴洗耳水治疗能够控制耳炎的临床症状，以及细菌和真菌过度生长。 结论和临床重要性-微生物过度生长引起的轻度耳炎病例，可以使用外部抗菌剂和抗炎药进行治疗，而不需要抗生素和/或抗真菌药联合治疗.A otite externa canina (OE) é uma doença comum caracterizada por inflamação do tecido epitelial do conduto auditivo externo. Infecções secundárias são frequentes, e Malassezia pachydermatis e Staphylococcus pseudintermediussão rotineiramente isolados e tratados com fórmulas antifúngicas e antibacterianas. HIPÓTESE/OBJETIVOS: Analisar o microbioma da orelha com otite antes e após o tratamento com prednisolona com romã ou antimicrobianos.15 cães com OE não purulenta. MÉTODOS E MATERIAIS: Um estudo duplo-cego, multicêntrico, randomizado, placebo-controle (1:1) de 30 dias foi realizado em 15 cães com otite externa não purulenta, seguindo dois tratamentos tópicos diferentes (prednisolona com romã versus prednisolona com antibiótico e antifúngico). Nos dias (D)0, D15 e D30, amostras séricas e otológicas foram coletadas, e realizou-se exames clínicos e análise de microbioma (bacteriano e fúngico). Os resultados foram comparados a escores clínicos validados para otite para avaliar a eficácia de ambos os tratamentos.Nove famílias bacterianas e quadro fúngicas foram detectadas durante os três tempos de coleta testados. Um aumento na diversidade (Shannon index) e composição fúngica foi a alteração mais significativa em ambos os grupos de tratamento (P ≤ 0.05). O limpador a base de prednisolona e romã foi capaz de controlar os sinais clínicos de otite, assim como o supercrescimento de bactérias e fungos. CONCLUSÕES E IMPORTÂNCIA CLÍNICA: Casos leves de otite associados a supercrescimento microbiano podem ser manejados com formulações tópicas antissépticas e anti-inflamatórias sem a necessidade de utilizar compostos antifúngicos e antibacterianos.

The concept that ruminant mammary gland quarters are anatomically and physiologically unrelated has been recently challenged by immunological evidence. How this interdependence reflects on individual quarter milk microbiota is unknown. The aim of the present study was to cover this gap by investigating the interdependence of quarters among the same mammary gland at the milk microbiota level using next-generation sequencing of the V4-16S rRNA gene. A total of 52 samples were included in this study and classified as healthy or affected by subclinical mastitis. Extraction of DNA, amplification of the V4-16S rRNA gene, and sequencing using Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, Waltham, MA) were carried out. We found that the intra-individual variability was lower than the inter-individual one. The present findings further support at milk microbiota level the hypothesis of the interdependence of quarters, as previously demonstrated following immunological studies, suggesting that individual factors (e.g., immunity, genetics) may have a role in modulating milk microbiota.

Altres ajuts: PRC/IZS/ME/10/13 RC. AC Water buffalo mastitis represents a major issue in terms of animal health, cost of therapy, premature culling and decreased milk yeld. The emergence of antibiotic resistance has led to investigate strategies to avoid or reduce antibiotics' based therapies, in particular during subclinical mastitis. The use of Generally Regarded As Safe bacteria (GRAS) such as Lactobacillus rhamnosus to restore the unbalance in mammary gland microbiota could provide potential corrective measures. The aim of this study was to investigate the changes in milk microbiota after the intramammary treatment with inactivated cultures of Lactobacillus rhamnosus of mammary gland quarters naturally affected by subclinical mastitis as compared to antibiotic therapy.A number of 43 quarters affected by subclinical mastitis with no signs of clinical inflammation and aerobic culture positive for pathogens were included in the study. The experimental design was as follows: 11 quarters were treated with antibiotics, 15 with inactivated cultures of Lactobacillus rhmnosus and 17 with PBS as negative control, by means of intrammary injection. Samples were collected at eight time points, pre- (T-29, T-21, T-15, T-7, T0 days) and post- treatment (T1, T2, and T6 days). Microbiological culture and Somatic Cell Count (SCC) were perfomed on all the samples, and microbiota was determined on milk samples collected at T0 and T6 by amplifying the V4 region of 16S rRNA gene by PCR and sequencing using next generation sequencing technique. Treatment with Lactobacillus rhamnosus elicited a strong chemotactic response, as determined by a significant increase of leukocytes in milk, but did not change the microbiological culture results of the treated quarters. For what concerns the analysis of the microbiota, the treatment with Lactobacillus rhamnosus induced the modification in relative abundance of some genera such as Pseudomonas and 5-7N15. As expected, antibiotic treatment caused major changes in microbiota structure with an increase of Methylobacterium relative abundance. No changes were detected after PBS treatment. In conclusion, the present findings demonstrated that the in vivo intrammmary treatment with Lactobacillus rhamnosus has a transient pro-inflammatory activity by increasing SCC and is capable to modify the microbiota of milk after six days from inoculation, albeit slightly, even when the bacterial cultures were heat inactivated. Further studies are necessary to assess the potential use of this GRAS as supportive therapy against mastitis.

Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples are prone to contain DNA from other sources (e.g. host or environment). The usual approach is sequencing short regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. To achieve an increased taxonomic resolution, we aim to develop long-amplicon PCR-based approaches using Nanopore sequencing. We assessed two different genetic markers: the full-length 16S rRNA (~1,500 bp) and the 16S-ITS-23S region from the rrn operon (4,300 bp). Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities and two pools of low-biomass samples (dog skin). Nanopore sequencing was performed on MinION™ using the 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using EPI2ME or Minimap2 with rrn database. Consensus sequences of the 16S-ITS-23S genetic marker were obtained using canu. Results: The full-length 16S rRNA and the 16S-ITS-23S region of the rrn operon were used to retrieve the microbiota composition of the samples at the genus and species level. For the Staphylococcus pseudintermedius isolate, the amplicons were assigned to the correct bacterial species in ~98% of the cases with the16S-ITS-23S genetic marker, and in ~68%, with the 16S rRNA gene when using EPI2ME. Using mock communities, we found that the full-length 16S rRNA gene represented better the abundances of a microbial community; whereas, 16S-ITS-23S obtained better resolution at the species level. Finally, we characterized low-biomass skin microbiota samples and detected species with an environmental origin. Conclusions: Both full-length 16S rRNA and the 16S-ITS-23S of the rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, targeting the 16S-ITS-23S of the rrn operon would be the best choice.

Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples often contain DNA from other sources, such as the host or the environment. The usual approach is sequencing specific hypervariable regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. Here, we aim to assess long-amplicon PCR-based approaches for assigning taxonomy at the genus and species level. We use Nanopore sequencing with two different markers: full-length 16S rRNA (~1,500 bp) and the whole rrn operon (16S rRNA–ITS–23S rRNA; 4,500 bp). Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities (HM-783D, Bei Resources; D6306, ZymoBIOMICS™) and two pools of low-biomass samples (dog skin from either the chin or dorsal back), using the MinION™ sequencer 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using the WIMP workflow on EPI2ME or Minimap2 software with rrn database. Results: The full-length 16S rRNA and the rrn operon were used to retrieve the microbiota composition at the genus and species level from the bacterial isolate, mock communities and complex skin samples. For the Staphylococcus pseudintermedius isolate, when using EPI2ME, the amplicons were assigned to the correct bacterial species in ~98% of the cases with the rrn operon marker, and in ~68% of the cases with the 16S rRNA gene. In both skin microbiota samples, we detected many species with an environmental origin. In chin, we found different Pseudomonas species in high abundance, whereas in dorsal skin there were more taxa with lower abundances. Conclusions: Both full-length 16S rRNA and the rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, using the rrn operon would be the best choice.

Myobia sp. and Demodex sp. are two skin mites that infest mice, particularly immunodeficient or transgenic lab mice. In the present study, wild house mice from five localities from the Barcelona Roberstonian system were analysed in order to detect skin mites and compare their prevalence between standard (2n = 40) and Robertsonian mice (2n > 40). We found and identified skin mites through real-time qPCR by comparing sequences from the mitochondrial 16S rRNA and the nuclear 18S rRNA genes since no sequences are available so far using the mitochondrial gene. Fourteen positive samples were identified as Myobia musculi except for a deletion of 296 bp out to 465 bp sequenced, and one sample was identified as Demodex canis. Sampling one body site, the mite prevalence in standard and Robertsonian mice was 0 and 26%, respectively. The malfunction of the immune system elicits an overgrowth of skin mites and consequently leads to diseases such as canine demodicosis in dogs or rosacea in humans. In immunosuppressed mice, the probability of developing demodicosis is higher than in healthy mice. Since six murine toll-like receptors (TLRs) are located in four chromosomes affected by Robertsonian fusions, we cannot dismiss that differences in mite prevalence could be the consequence of the interruption of TLR function. Although ecological and/or morphological factors cannot be disregarded to explain differences in mite prevalence, the detection of translocation breakpoints in TLR genes or the analysis of TLR gene expression are needed to elucidate how Robertsonian fusions affect the immune system in mice., Field work and karyotyping of the mice from the Barcelona Robertsonian system used in this study was funded by the Spanish Ministerio de Ciencia y Tecnología (project number CGL2007-62111 to JV) and Ministerio de Economía y Competitividad (project reference CGL2010-15243 to JV). Financial support for genetic analysis was provided by the “Servei Veterinari de Genètica Molecular” (SVGM).

Objectives The presence of Bartonella spp. was detected by polymerase chain reaction (PCR) in dogs from Spain with blood culture-negative endocarditis. The aim of this study is to add information about canine infectious endocarditis in Europe. Animals Thirty dogs with naturally occurring blood culture-negative endocarditis were examined from 2010 to 2017 at three veterinary referral hospitals, located in northwest, northeast, and southeast of Spain. Methods It is a retrospective study. Medical records were reviewed to extract relevant data. Frozen or paraffin-embedded cardiac valve tissue and/or ethylenediamine tetraacetic acid blood samples were evaluated by PCR for the presence of Bartonella DNA. Positive results were sequenced to confirm the species. Results Polymerase chain reaction was positive for eight out of 30 dogs included (26.6%). Bartonella rochalimae, Bartonella vinsonii subsp. berkhoffii, and Bartonella koehlerae were detected in valve tissue or blood. Conclusions Bartonella could be an important cause of blood culture-negative infectious endocarditis in dogs from Spain. The outcome for those dogs affected with Bartonella spp. was grave. Prompt empirical treatment with amoxicillin-clavulanate plus fluoroquinolones could be of value in cases of blood culture-negative endocarditis.

The most common strategy to assess microbiota is sequencing specific hypervariable regions of 16S rRNA gene using 2ndgeneration platforms (such as MiSeq or Ion Torrent PGM). Despite obtaining high-quality reads, many sequences fail to be classified at the genus or species levels due to their short length. This pitfall can be overcome sequencing the full-length 16S rRNA gene (1,500bp) by 3rdgeneration sequencers.We aimed to assess the performance of nanopore sequencing using MinION™on characterizing microbiota complex samples. First set-up step was performed using a staggered mock community (HM-783D). Then, we sequenced a pool of several dog skin microbiota samples previously sequenced by Ion Torrent PGM. Sequences obtained for full-length 16S rRNA with degenerated primers retrieved increased richness estimates at high taxonomic level (Bacteria and Archaea) that were missed with short-reads. Besides, we were able to obtain taxonomic assignments down to species level, although it was not always feasible due to: i) incomplete database; ii) primer set chosen; iii) low taxonomic resolution of 16S rRNA gene within some genera; and/or iv) sequencing errors. Nanopore sequencing of the full-length 16S rRNA gene using MinION™with 1D sequencing kit allowed us inferring microbiota composition of a complex microbial community to lower taxonomic levels than short-reads from 2ndgeneration sequencers.

Arrhythmias are frequent and can be caused by many diseases. Vector-borne diseases are increasing in importance as a cause of arrhythmias in veterinary medicine. The objective of the study was to evaluate dogs presenting arrhythmias of any aetiology and to assess the influence of polymerase chain reaction (PCR) status for vector-borne pathogens in the Mediterranean area of Spain. A total of 120 dogs presenting arrhythmias were prospectively selected, and PCR for several vector-borne pathogens (Leishmania infantum, Ehrlichia/Anaplasma spp., Babesia spp., Rickettsia spp., Mycoplasma spp., Hepatozoon canis and Bartonella spp.) were analysed in blood samples. Twenty-four (20 %) dogs had positive results for PCR. Found pathogens were Ehrlichia/Anaplasma spp. (n = 10, 8.3 %), Babesia spp. (n = 7, 5.8 %), Rickettsia spp. (n = 5, 4.2 %), Leishmania spp. (n = 4, 3.3 %), Mycoplasma spp. (n = 4, 3.3 %), and H. canis (n = 1, 0.8 %). No significant differences were found in relation to PCR status and type or cause of the arrhythmia. Although there is a clear association between some infectious diseases and cardiac diseases, this study could not find evidence of the role of infectious agents as triggers for arrhythmias in dogs. More studies are needed to further explore this association.

The identification of Demodex injai as a second Demodex species of dog opened new questions and challenges in the understanding on the Demodex-host relationships. In this paper, we describe the development of a conventional PCR technique based on published genome sequences of D. injai from GenBank that specifically detects DNA from D. injai. This technique amplifies a 238-bp fragment corresponding to a region of the mitochondrial 16S rDNA of D. injai. The PCR was positive in DNA samples obtained from mites identified morphologically as D. injai, which served as positive controls, as well as in samples from three cases of demodicosis associated with proliferation of mites identified as D. injai. Furthermore, the PCR was positive in 2 out of 19 healthy dogs. Samples of Demodex canis and Demodex folliculorum were consistently negative. Skin samples from seven dogs with generalized demodicosis caused by D. canis were all negative in the D. injai-specific PCR, demonstrating that in generalized canine demodicosis, mite proliferation is species-specific. This technique can be a useful tool in the diagnosis and in epidemiologic and pathogenic studies.