Isolation and molecular characterization of a novel relapsing fever group Borrelia from the white-eared opossum Didelphis albiventris in Brazil.

This study aimed to detect, isolate and to characterize by molecular methods a relapsing fever group (RFG) Borrelia in white-eared opossums (Didelphis albiventris) from Brazil. During 2015-2018, when opossums (Didelphis spp.) were captured in six municipalities of the state of São Paulo, Brazil, molecular analyses revealed the presence of a novel RFG Borrelia sp. in the blood of seven opossums (Didelphis albiventris), out of 142 sampled opossums (4.9% infection rate). All seven infected opossums were from a single location (Ribeirão Preto municipality). In a subsequent field study in Ribeirão Preto during 2021, two new opossums (D. albiventris) were captured, of which one contained borrelial DNA in its blood. Macerated tissues from this infected opossum were inoculated into laboratory animals (rodents and rabbits) and two big-eared opossums (Didelphis aurita), which had blood samples examined daily via dark-field microscopy. No spirochetes were visualized in the blood of the laboratory animals. Contrastingly, spirochetes were visualized in the blood of the two D. aurita opossums between 12 and 25 days after inoculation. Blood samples from these opossums were used for a multi-locus sequencing typing (MLST) based on six borrelial loci. Phylogenies inferred from MLST genes positioned the sequenced Borrelia genotype into the RFG borreliae clade basally to borreliae of the Asian-African group, forming a monophyletic group with another Brazilian isolate, "Candidatus B. caatinga". Based on this concatenated phylogenetic analysis, which supports that the new borrelial isolate corresponds to a putative new species, we propose the name "Candidatus Borrelia mimona".

Insights into the genetic architecture of Phytophthora capsici root rot resistance in chile pepper (Capsicum spp.) from multi-locus genome-wide association study.

BACKGROUND: Phytophthora root rot, a major constraint in chile pepper production worldwide, is caused by the soil-borne oomycete, Phytophthora capsici. This study aimed to detect significant regions in the Capsicum genome linked to Phytophthora root rot resistance using a panel consisting of 157 Capsicum spp. genotypes. Multi-locus genome wide association study (GWAS) was conducted using single nucleotide polymorphism (SNP) markers derived from genotyping-by-sequencing (GBS). Individual plants were separately inoculated with P. capsici isolates, 'PWB-185', 'PWB-186', and '6347', at the 4-8 leaf stage and were scored for disease symptoms up to 14-days post-inoculation. Disease scores were used to calculate disease parameters including disease severity index percentage, percent of resistant plants, area under disease progress curve, and estimated marginal means for each genotype. RESULTS: Most of the genotypes displayed root rot symptoms, whereas five accessions were completely resistant to all the isolates and displayed no symptoms of infection. A total of 55,117 SNP markers derived from GBS were used to perform multi-locus GWAS which identified 330 significant SNP markers associated with disease resistance. Of these, 56 SNP markers distributed across all the 12 chromosomes were common across the isolates, indicating association with more durable resistance. Candidate genes including nucleotide-binding site leucine-rich repeat (NBS-LRR), systemic acquired resistance (SAR8.2), and receptor-like kinase (RLKs), were identified within 0.5 Mb of the associated markers. CONCLUSIONS: Results will be used to improve resistance to Phytophthora root rot in chile pepper by the development of Kompetitive allele-specific markers (KASP®) for marker validation, genomewide selection, and marker-assisted breeding.

The structure of associations: Method insights from analyzing 28 clinical isolates of Cryptococcus neoformans.

Clinical isolates of a fungal pathogen from a single region or country often exhibit structural clonality or phylogenetic clustering at the sequence or MLST level; such population structure can persist also in larger samples. In efforts to improve causal understanding of pathogenesis at the molecular level, genome-wide association screening methods initially designed for other kingdoms have been applied to fungi. The example of a Colombian dataset of 28 clinical Cryptococcus neoformans VNI isolates indicates where the output from standard pipelines may need to be analyzed in new ways in order to efficiently extract hypotheses for experiments from fungal genotype-phenotype data.

Collections of clinical isolates of a human fungal pathogen can consist of clusters of genetically similar isolates. Such clustering complicates the screening for genetic associations with clinically relevant traits. We propose new methods, illustrating them for the fungus causing cryptococcosis.

Genome-wide association study for resistance to Pseudomonas syringae pv. garcae in Coffea arabica.

Bacteria halo blight (BHB), a coffee plant disease caused by Pseudomonas syringae pv. garcae, has been gaining importance in producing mountain regions and mild temperatures areas as well as in coffee nurseries. Most Coffea arabica cultivars are susceptible to this disease. In contrast, a great source of genetic diversity and resistance to BHB are found in C. arabica Ethiopian accessions. Aiming to identify quantitative trait nucleotides (QTNs) associated with resistance to BHB and the influence of these genomic regions during the domestication of C. arabica, we conducted an analysis of population structure and a Genome-Wide Association Study (GWAS). For this, we used genotyping by sequencing (GBS) and phenotyping for resistance to BHB of a panel with 120 C. arabica Ethiopian accessions from a historical FAO collection, 11 C. arabica cultivars, and the BA-10 genotype. Population structure analysis based on single-nucleotide polymorphisms (SNPs) markers showed that the 132 accessions are divided into 3 clusters: most wild Ethiopian accessions, domesticated Ethiopian accessions, and cultivars. GWAS, using the single-locus model MLM and the multi-locus models mrMLM, FASTmrMLM, FASTmrEMMA, and ISIS EM-BLASSO, identified 11 QTNs associated with resistance to BHB. Among these QTNs, the four with the highest values of association for resistance to BHB are linked to g000 (Chr_0_434_435) and g010741 genes, which are predicted to encode a serine/threonine-kinase protein and a nucleotide binding site leucine-rich repeat (NBS-LRR), respectively. These genes displayed a similar transcriptional downregulation profile in a C. arabica susceptible cultivar and in a C. arabica cultivar with quantitative resistance, when infected with P. syringae pv. garcae. However, peaks of upregulation were observed in a C. arabica cultivar with qualitative resistance, for both genes. Our results provide SNPs that have potential for application in Marker Assisted Selection (MAS) and expand our understanding about the complex genetic control of the resistance to BHB in C. arabica. In addition, the findings contribute to increasing understanding of the C. arabica domestication history.

Multi-locus DNA methylation analysis of imprinted genes in cattle from somatic cell nuclear transfer.

Multi-locus methylation defects (MLMDs) in imprinted loci have been reported in Beckwith-Wiedemann Syndrome (BWS). Large offspring syndrome (LOS), a phenotypic subgroup of abnormal offspring syndrome (AOS), is considered a molecular and phenotypic model for BWS. Both LOS and BWS have presented epigenetic defects in some common imprinted loci. In this study, methylation-specific restriction digestion assay - quantitative PCR was used to analyze the DNA methylation pattern in differentially methylated regions (DMRs) of the H19 (H19-DMR), KCNQ1OT1 (KvDMR1) and PEG1/MEST (PEG1-DMR) genes in bovine clone tissues from calves that did not survive after birth. Individual and tissue-specific changes in DNA methylation levels in the bovine KvDMR1, H19-DMR, and PEG1-DMR were observed. In contrast to what has been reported in the literature on BWS and AOS/LOS, the KvDMR1 showed gain (GOM) and loss (LOM) of DNA methylation. LOM and GOM events were found in the DMRs studied in animals produced by the same nucleus donor cell line. This is the first report of epimutations in the PEG1-DMR and GOM at the KvDMR1 found in bovine clones. The findings showed that epigenetic modification in imprinted loci in cloned cattle occurred in a multi-locus pattern similar to that seen in human imprinting disorders. Other multi-locus analyzes must be done to elucidate the MLMD pattern in AOS in bovine clones.

Genomic characterization, antimicrobial resistance profiles, enterotoxin, and biofilm production of methicillin-resistant Staphylococcus aureus isolated from clinical and animal products origins in Eastern Turkey

ABSTRACT: Staphylococcus aureus is an opportunistic and ubiquitous pathogen found in the skin, nares, and mucosal membranes of mammals. Increasing resistance to antimicrobials including methicillin has become an important public concern. One hundred and eight (108) S. aureus strains isolated from a total of 572 clinical and animal products samples, were investigated for their biofilm capability, methicillin resistance, enterotoxin genes, and genetic diversity. Although only one strain isolated from raw retail was found as a strong biofilm producer, the percentage of antimicrobial resistance pattern was relatively higher. 17.59% of S. aureus strains tested in this study were resistant to cefoxitin and identified as methicillin-resistant S. aureus (MRSA) isolates. mecA and mecC harboring S. aureus strains were detected at a rate of 2.79% and 0.93%, respectively. In addition, staphylococcal enterotoxin genes including Sea, Seb, Sec, and Sed genes were found to be 18.5%, 32.4%, 6.5% and 3.7%, respectively. The phylogenetic relationship among the isolates showed relationship between joint calf and cow milk isolates. Multi locus sequence typing (MLST) revealed three different sequence types (STs) including ST84, ST829, and ST6238. These findings highlight the development and spread of MRSA strains with zoonotic potential in animals and the food chain throughout the world.

RESUMO: Staphylococcus aureus é um patógeno dúctil e ubíquo encontrado na pele, narinas e membranas mucosas de mamíferos. O aumento da resistência aos antimicrobianos, incluindo a meticilina, tornou-se uma importante preocupação pública. Cento e oito (108) cepas de S. aureus isoladas de um total de 572 amostras clínicas e de produtos animais foram investigadas por sua capacidade de biofilme, resistência à meticilina, genes de enterotoxinas e diversidade genética. Embora apenas uma cepa isolada do cru tenha sido encontrada como forte produtora de biofilme, a porcentagem do padrão de resistência antimicrobiana foi relativamente maior. Parte das cepas (17,59%) de S. aureus testadas neste estudo eram resistentes à cefoxitina e identificadas como isolados de MRSA. mecA e mecC abrigando cepas de S. aureus foram detectados a uma taxa de 2,79% e 0,93%, respectivamente. Além disso, verificou-se que os genes da enterotoxina estafilocócica, incluindo os genes Sea, Seb, Sec e Sed, eram 18,5%, 32,4%, 6,5% e 3,7%, respectivamente. A relação filogenética entre os isolados mostrou relação entre os isolados de bezerro e leite de vaca. A tipagem de sequência multiloco (MLST) revelou três tipos de sequência diferentes (STs), incluindo ST84, ST829 e ST6238. Essas descobertas destacam o desenvolvimento e a disseminação de cepas de MRSA com potencial zoonótico em animais e na cadeia alimentar em todo o mundo.

Genomic characterization, antimicrobial resistance profiles, enterotoxin, and biofilm production of methicillin-resistant Staphylococcus aureus isolated from clinical and animal products origins in Eastern Turkey / Caracterização genômica perfis de resistência antimicrobiana, produção de enterotoxinas e biofilme Staphylococcus aureus resistente à meticilina isolado de origem clínica e de produtos de origem animal no leste da Turquia

Staphylococcus aureus is an opportunistic and ubiquitous pathogen found in the skin, nares, and mucosal membranes of mammals. Increasing resistance to antimicrobials including methicillin has become an important public concern. One hundred and eight (108) S. aureus strains isolated from a total of 572 clinical and animal products samples, were investigated for their biofilm capability, methicillin resistance, enterotoxin genes, and genetic diversity. Although only one strain isolated from raw retail was found as a strong biofilm producer, the percentage of antimicrobial resistance pattern was relatively higher. 17.59% of S. aureus strains tested in this study were resistant to cefoxitin and identified as methicillin-resistant S. aureus (MRSA) isolates. mecA and mecC harboring S. aureus strains were detected at a rate of 2.79% and 0.93%, respectively. In addition, staphylococcal enterotoxin genes including Sea, Seb, Sec, and Sed genes were found to be 18.5%, 32.4%, 6.5% and 3.7%, respectively. The phylogenetic relationship among the isolates showed relationship between joint calf and cow milk isolates. Multi locus sequence typing (MLST) revealed three different sequence types (STs) including ST84, ST829, and ST6238. These findings highlight the development and spread of MRSA strains with zoonotic potential in animals and the food chain throughout the world.

Staphylococcus aureus é um patógeno dúctil e ubíquo encontrado na pele, narinas e membranas mucosas de mamíferos. O aumento da resistência aos antimicrobianos, incluindo a meticilina, tornou-se uma importante preocupação pública. Cento e oito (108) cepas de S. aureus isoladas de um total de 572 amostras clínicas e de produtos animais foram investigadas por sua capacidade de biofilme, resistência à meticilina, genes de enterotoxinas e diversidade genética. Embora apenas uma cepa isolada do cru tenha sido encontrada como forte produtora de biofilme, a porcentagem do padrão de resistência antimicrobiana foi relativamente maior. Parte das cepas (17,59%) de S. aureus testadas neste estudo eram resistentes à cefoxitina e identificadas como isolados de MRSA. mecA e mecC abrigando cepas de S. aureus foram detectados a uma taxa de 2,79% e 0,93%, respectivamente. Além disso, verificou-se que os genes da enterotoxina estafilocócica, incluindo os genes Sea, Seb, Sec e Sed, eram 18,5%, 32,4%, 6,5% e 3,7%, respectivamente. A relação filogenética entre os isolados mostrou relação entre os isolados de bezerro e leite de vaca. A tipagem de sequência multiloco (MLST) revelou três tipos de sequência diferentes (STs), incluindo ST84, ST829 e ST6238. Essas descobertas destacam o desenvolvimento e a disseminação de cepas de MRSA com potencial zoonótico em animais e na cadeia alimentar em todo o mundo.

Description of the First Four Species of the Genus Pseudogymnoascus From Antarctica.

The genus Pseudogymnoascus represents a diverse group of fungi widely distributed in different cold regions on Earth. Our current knowledge of the species of Pseudogymnoascus is still very limited. Currently, there are only 15 accepted species of Pseudogymnoascus that have been isolated from different environments in the Northern Hemisphere. In contrast, species of Pseudogymnoascus from the Southern Hemisphere have not yet been described. In this work, we characterized four fungal strains obtained from Antarctic marine sponges. Based on multilocus phylogenetic analyses and morphological characterizations we determined that these strains are new species, for which the names Pseudogymnoascus antarcticus sp. nov., Pseudogymnoascus australis sp. nov., Pseudogymnoascus griseus sp. nov., and Pseudogymnoascus lanuginosus sp. nov. are proposed. Phylogenetic analyses indicate that the new species form distinct lineages separated from other species of Pseudogymnoascus with strong support. The new species do not form sexual structures and differ from the currently known species mainly in the shape and size of their conidia, the presence of chains of arthroconidia, and the appearance of their colonies. This is the first report of new species of Pseudogymnoascus not only from Antarctica but also from the Southern Hemisphere.

Genomic surveillance of invasive Streptococcus pneumoniae isolates in the period pre-PCV10 and post-PCV10 introduction in Brazil.

In 2010, Brazil introduced the 10-valent pneumococcal conjugate vaccine (PCV10) into the national children's immunization programme. This study describes the genetic characteristics of invasive Streptococcus pneumoniae isolates before and after PCV10 introduction. A subset of 466 [pre-PCV10 (2008-2009): n=232, post-PCV10 (2012-2013): n=234;<5 years old: n=310, ≥5 years old: n=156] pneumococcal isolates, collected through national laboratory surveillance, were whole-genome sequenced (WGS) to determine serotype, pilus locus, antimicrobial resistance and genetic lineages. Following PCV10 introduction, in the <5 years age group, non-vaccine serotypes (NVT) serotype 3 and serotype 19A were the most frequent, and serotypes 12F, 8 and 9 N in the ≥5 years old group. The study identified 65 Global Pneumococcal Sequence Clusters (GPSCs): 49 (88 %) were GPSCs previously described and 16 (12 %) were Brazilian clusters. In total, 36 GPSCs (55 %) were NVT lineages, 18 (28 %) vaccine serotypes (VT) and 11 (17 %) were both VT and NVT lineages. In both sampling periods, the most frequent lineage was GPSC6 (CC156, serotypes 14/9V). In the <5 years old group, a decrease in penicillin (P=0.0123) and cotrimoxazole (P<0.0001) resistance and an increase in tetracycline (P=0.019) were observed. Penicillin nonsusceptibility was predicted in 40 % of the isolates; 127 PBP combinations were identified (51 predicted MIC≥0.125 mg l-1); cotrimoxazole (folA and/or folP alterations), macrolide (mef and/or ermB) and tetracycline (tetM, tetO or tetS/M) resistance were predicted in 63, 13 and 21.6 % of pneumococci studied, respectively. The main lineages associated with multidrug resistance in the post-PCV10 period were composed of NVT, GPSC1 (CC320, serotype 19A), and GPSC47 (ST386, serotype 6C). The study provides a baseline for future comparisons and identified important NVT lineages in the post-PCV10 period in Brazil.

Multi-locus imprinting disturbances of Beckwith-Wiedemann and Large offspring syndrome/Abnormal offspring syndrome: A brief review.

In vitro fertilization and somatic cell nuclear transfer are assisted reproduction technologies commonly used in humans and cattle, respectively. Despite advances in these technologies, molecular failures can occur, increasing the chance of the onset of imprinting disorders in the offspring. Large offspring syndrome/abnormal offspring syndrome (LOS/AOS) has been described in cattle and has features such as hypergrowth, malformation of organs, and skeletal and placental defects. In humans, Beckwith-Wiedemann syndrome (BWS) has phenotypic characteristics similar to those found in LOS/AOS. In both syndromes, disruption of genomic imprinting associated with loss of parental-specific expression and parental-specific epigenetic marks is involved in the molecular etiology. Changes in the imprinting pattern of these genes lead to loss of imprinting (LOI) due to gain or loss of methylation, inducing the emergence of these syndromes. Several studies have reported locus-specific alterations in these syndromes, such as hypomethylation in imprinting control region 2 (KvDMR1) in BWS and LOS/AOS. These LOI events can occur at multiple imprinted loci in the same affected individual, which are called multi-locus methylation defect (MLMD) events. Although the bovine species has been proposed as a developmental model for human imprinting disorders, there is little information on bovine imprinted genes in the literature, even the correlation of epimutation data with clinical characteristics. In this study, we performed a systematic review of all the multi-locus LOI events described in human BWS and LOS/AOS, in order to determine in which imprinted genes the largest changes in the pattern of DNA methylation and expression occur, helping to fill gaps for a better understanding of the etiology of both syndromes.

Phylogenomic analysis for Campylobacter fetus ocurring in Argentina.

BACKGROUND AND AIM: Campylobacter fetus is one of the most important pathogens that severely affects livestock industry worldwide. C. fetus mediated bovine genital campylobacteriosis infection in cattle has been associated with significant economic losses in livestock production in the Pampas region, the most productive area of Argentina. The present study aimed to establish the genomic relationships between C. fetus strains, isolated from the Pampas region, at local and global levels. The study also explored the utility of multi-locus sequence typing (MLST) as a typing technique for C. fetus. MATERIALS AND METHODS: For pangenome and phylogenetic analysis, whole genome sequences for 34 C. fetus strains, isolated from cattle in Argentina were downloaded from GenBank. A local maximum likelihood (ML) tree was constructed and linked to a Microreact project. In silico analysis based on MLST was used to obtain information regarding sequence type (ST) for each strain. For global phylogenetic analysis, a core genome ML-tree was constructed using genomic dataset for 265 C. fetus strains, isolated from various sources obtained from 20 countries. RESULTS: The local core genome phylogenetic tree analysis described the presence of two major clusters (A and B) and one minor cluster (C). The occurrence of 82% of the strains in these three clusters suggested a clonal population structure for C. fetus. The MLST analysis for the local strains revealed that 31 strains were ST4 type and one strain was ST5 type. In addition, a new variant was identified that was assigned a novel ST, ST70. In the present case, ST4 was homogenously distributed across all the regions and clusters. The global analysis showed that most of the local strains clustered in the phylogenetic groups that comprised exclusively of the strains isolated from Argentina. Interestingly, three strains showed a close genetic relationship with bovine strains obtained from Uruguay and Brazil. The ST5 strain grouped in a distant cluster, with strains obtained from different sources from various geographic locations worldwide. Two local strains clustered in a phylogenetic group comprising intercontinental Campylobacter fetus venerealis strains. CONCLUSION: The results of the study suggested active movement of animals, probably due to economic trade between different regions of the country as well as with neighboring countries. MLST results were partially concordant with phylogenetic analysis. Thus, this method did not qualify as a reliable subtyping method to assess C. fetus diversity in Argentina. The present study provided a basic platform to conduct future research on C. fetus, both at local and international levels.

Molecular typing of multi-drug resistant Candida albicans isolated from the Segamat community, Malaysia.

In the past decade, researchers have focused on the emergence of drug resistance in fungal pathogens such as Candida albicans, also considered as pathobionts that occur harmlessly in the human body but could potentially be triggered to cause diseases. The increasing rate of antifungal resistance in commensal gut fungi is alarming and should be further investigated. Here, we report seven novel MLST (Multi Locus Sequence Typing) genotypes of multi-drug resistant C. albicans isolates obtained from participants of a community study in Segamat, a district in the state of Johor, Malaysia. A total of eight C. albicans were isolated from four individuals, which were found to express high resistance against fluconazole, itraconazole, voriconazole and 5-fluorocytosine antifungals. MLST was performed to assess the clonal relatedness of these drug resistant isolates among themselves and against other strains isolated from other geographical regions. The novel MLST C. albicans sequence types suggest significant genetic changes compared to previous genotypes.

Genetic dissection of flowering time in flax (Linum usitatissimum L.) through single- and multi-locus genome-wide association studies.

In a rapidly changing climate, flowering time (FL) adaptation is important to maximize seed yield in flax (Linum usitatissimum L.). However, our understanding of the genetic mechanism underlying FL in this multipurpose crop remains limited. With the aim of dissecting the genetic architecture of FL in flax, a genome-wide association study (GWAS) was performed on 200 accessions of the flax core collection evaluated in four environments. Two single-locus and six multi-locus models were applied using 70,935 curated single nucleotide polymorphism (SNP) markers. A total of 40 quantitative trait nucleotides (QTNs) associated with 27 quantitative trait loci (QTL) were identified in at least two environments. The number of QTL with positive-effect alleles in accessions was significantly correlated with FL (r = 0.77 to 0.82), indicating principally additive gene actions. Nine QTL were significant in at least three of the four environments accounting for 3.06-14.71% of FL variation. These stable QTL spanned regions that harbored 27 Arabidopsis thaliana and Oryza sativa FL-related orthologous genes including FLOWERING LOCUS T (Lus10013532), FLOWERING LOCUS D (Lus10028817), transcriptional regulator SUPERMAN (Lus10021215), and gibberellin 2-beta-dioxygenase 2 (Lus10037816). In silico gene expression analysis of the 27 FL candidate gene orthologous suggested that they might play roles in the transition from vegetative to reproductive phase, flower development and fertilization. Our results provide new insights into the QTL architecture of flowering time in flax, identify potential candidate genes for further studies, and demonstrate the effectiveness of combining different GWAS models for the genetic dissection of complex traits.

Genetic diversity of Mycobacterium avium sp. paratuberculosis by mycobacterial interspersed repetitive Unit-Variable number tandem repeat and multi-locus short-sequence repeat one-sentence summary: Genetic diversity of Mycobacterium avium sp. paratuberculosis isolates.

Background: Paratuberculosis is an enteric disease caused by Mycobacterium avium sp. paratuberculosis (MAP) that affects mainly ruminant producing losses to the livestock industry. Many molecular epidemiological methods have been used to discriminate MAP isolates. Method: The aim of this study was to describe the genetic diversity of the Argentinean MAP isolates using a combination of two molecular systems, the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) ("automated and "non-automated") and the multi-locus short-sequence repeat (MLSSR) system. Results: Thirty-two isolates were identified as MAP of C type by IS900 polymerase chain reaction (PCA) and IS1311 PCA-restriction enzyme analysis. The main patterns found by both MIRU-VNTR systems were INMV1 (54.5%), INMV2 (24.2%) and INMV11 (9.1%). The INMV5, INMV8 and INMV16 were represented with one isolate each (3.0%). Only 4 MIRU-VNTR loci were polymorphic. Conclusion: Those isolates sharing the same INMV patterns were analyzed by MLSSR, being locus 2 the most polymorphic one showing isolates with 9, 10, 11, and more than 11 "G" repeats. Besides, the global discriminatory power among isolates could be increased using both techniques. Based on these results, a short version of the "automated" MIRU-VNTR could be used as a screening tool to group isolates genetically related and subsequently perform the SSR using locus 2 on those isolates sharing the same INMV pattern.

First report of Colletotrichum tropicale causing anthracnose on Passiflora edulis in Brazil.

Brazil is the world's largest producer and consumer of yellow passion fruit (Passiflora edulis f. flavicarpa), mainly for the manufacture of concentrate and frozen juice as well as for fresh consumption (Faleiro et al. 2005). Between June and July 2018, passion fruit plants with symptoms of anthracnose were observed in commercial planting in the municipality of Coruripe (20 ha), northeastern state of Alagoas, Brazil. Approximately 70% of the plants showed leaves with relatively large, watery, circular spots that affected 30% of the leaf surface. Small fragments taken from the transition region of symptomatic tissue were superficially disinfected in 70% ethanol for 30 s and in 1% NaClO for 1 min, rinsed in sterile distilled water (SDW), dried on filter paper, plated on potato dextrose agar (PDA-Kasvi) incubated at 25°C under white light and 12 h photoperiod, for 3 days. Two isolates were obtained and deposited in the Collection of Phytopathogens at the Universidade Federal de Alagoas (COUFAL0281 and COUFAL0282). To identify the isolates, partial sequences of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-tubulin (TUB2) genes and of the rDNA-ITS (ITS) region were amplified by PCR. The partial sequences were deposited at GenBank (MT299339, MT334694, MT310553, MT299340, MT334695 and MT310554). Based on the BLASTn analysis, sequences of the PCR products showed high nucleotide similarity with sequences of the species C. tropicale (CBS 124949/ex-type and ICMP 18672), for GAPDH (98.94% and 100%), TUB2 (99 and 100%) genes and ITS (100%). This result was also confirmed in the phylogenetic tree of Bayesian Inference assembled with concatenated data (GAPDH, TUB2 and ITS). The colonies of the isolates were white with a white reverse, with dense mycelium, and mean growth rate of 7.54 mm/day, after 7 days on PDA medium at 25° C. Conidia were subcylindrical with rounded ends, hyaline, smooth walls and measured 13.63-20.59µm (= 17.54µm; n= 50) in length and 4.40-7.93 µm (= 5.88 µm; n= 50) in width. Appressoria were melanized, subglobose, irregular and measured 7.44 - 18.57 µm (= 10.04 µm; n= 50) in length and 5.49-10.16 µm (= 7.66 µm; n= 50) in width. These morphological characteristics were consistent with those described for Colletotrichum tropicale E.I. Rojas, S.A. Rehner & Samuels (Rojas et al. 2010). To confirm pathogenicity, 30 µL of a 106 conidia/mL sterile distilled water (SDW) conidia suspension, together with a drop of 20% Tween were deposited on the adaxial surface of passion fruit leaves wounded with a sterile needle, with four repetitions. The control consisted of leaves inoculated only with SDW. The leaves were placed in a plastic Gerbox box with sterilized filter paper moistened with SDW and maintained in a Biochemistry Oxygen Demand (BOD) incubator stove at 25 ºC and photoperiod of 12 h. After 7 days, typical anthracnose symptoms were observed on inoculated leaves. The pathogen was re-isolated and confirmed by morphological characterization, according to Koch's postulates. No symptoms were observed in the negative control. The occurrence of this species has been frequently reported in several other crops grown in northeastern Brazil (Silva et al. 2017; Veloso et al. 2018; Vieira et al. 2018; Costa et al. 2019). Additionally, many of these crops are grown in close proximity to the passion fruit orchards, thus favoring pathogen movement between hosts, probably, due to the anthropic influence, circulation of animals and insects, as well as wind driven rain splashes. However, this is first report of C. tropicale in Passiflora edulis in the world.

Investigation of Genetic Diversity of Pasteurella multocida Isolated from Diseased Poultry in Korea

ABSTRACT Pasteurella multocida causes fowl cholera which is an economically important disease in poultry industries around the world. In this study, we analyzed the capsular genotype, lipopolysaccharide (LPS) genotype, virulence-associated genes (VAGs) patterns, antimicrobial resistance and genetic diversity in a total of 9 P. multocida isolates from poultry with fowl cholera between 2014 and 2019 in Korea. When combining the capsular types with the LPS genotypes, two isolates of the 9 isolates were A:L3, and the others were non-typeable (NT): L3. Of the 23 VAGs, all the isolates harbored ptfA, fimA, hsf-1, hsf-2, pfhA, exbB, exbD, tonB, hgbA, hgbB, fur, sodA, sodC, pmHAS, ompA, ompH, oma87, plpB, psl, and nanH, whereas toxA gene was not detected in any of the 9 isolates. In addition, among the 11 antimicrobials, most of the isolates except for one isolate resistant to florfenicol, exhibited susceptibility to all the antimicrobials. Multi-locus sequence typing (MLST) analysis revealed 5 different sequence types (ST): ST8, ST351, ST352, ST353, and ST368. The ST351, ST352, ST353, and ST368 were identified for the first time in this study, and ST352 and ST353 isolates were largely prevalent nationwide. These STs isolates should be monitored continuously because in some cases, ST352 and ST353 isolates demonstrated high mortality rates. Although only limited numbers of isolates have been analyzed, our findings provide overall characteristics and epidemiological information of the P. multocida strains recently prevalent in Korea.

New insights on evolutionary aspects of Pythium insidiosum and other peronosporaleans.

BACKGROUND: The evolution of pathogenic mechanisms is a major challenge, which requires a thorough comprehension of the phylogenetic relationships of pathogens. Peronosporaleans encompasses a heterogeneous group of oomycetes that includes some animal/human pathogens, like Pythium insidiosum. OBJECTIVE: We analysed here the phylogenetic positioning and other evolutionary aspects related to this species and other peronosporaleans, using a multi-locus approach with one mitochondrial and three nuclear genes. METHODOLOGY: Phylogenetic patterns of 55 oomycetes were inferred by maximum likelihood and Bayesian analysis, and a relaxed molecular clock method was applied to infer the divergence time of some peronosporaleans branches. RESULTS: Pythium insidiosum was monophyletic with a major and polytomous clade of American isolates; however, Pythium spp. was found to be paraphyletic with Phytopythium sp. and Phytophthora spp. In general, peronosporaleans subdivided into four lineages, one of which evidenced a close relationship of P insidiosum, P aphanidermatum and P arrhenomanes. This lineage diverged about 63 million years ago (Mya), whereas P insidiosum diversified at approximately 24 Mya. The divergence of American and Thai isolates seems to have occurred at approximately 17 Mya, with further American diversification at 2.4 Mya. CONCLUSION: Overall, this study clarifies the phylogenetic relationships of P insidiosum regarding other peronosporaleans in a multi-locus perspective, despite previous claims that phylogenomic analyses are needed to accurately infer the patterns and processes related to the evolution of different lineages in this group. Additionally, this is the first time that a molecular clock was applied to study the evolution of P insidiosum.

Clonality of Mycoplasma hyopneumoniae in swine farms from Brazil.

Mycoplasma hyopneumoniae causes enzootic pneumonia (EP) in swine, a disease related to high economic losses in production systems. Epidemiological spread of M. hyopneumoniae clones was studied by multi-locus sequence typing (MLST) in several swine production regions but so far not in South America. Using MLST, we have therefore investigated M. hyopneumoniae clones circulating in farms from three main swine production regions in Brazil. Porcine lungs samples were collected between 2015 and 2016 in farms with EP outbreaks. Three geographically distant regions were selected, and 67 M. hyopneumoniae positive samples, each one from a different farm, were included in the study. The occurrence of five sequence types (ST) was demonstrated and the majority of the samples were identified as ST-69 (n = 60; 89.5%), followed by ST-70 (n = 3; 4.5%), ST-123 (n = 2; 3%), ST-124 (n = 1; 1.5%) and ST-127 (n = 1; 1.5%). There was no association of any specific ST with region or production system. The five STs were all new ones, probably representing unique Brazilian clones. ST-69 and ST-70 on one side and ST-123 and ST-124 on the other side are phylogenetically close, while ST-127 is singleton. In conclusion, our results showed a low variability and high clonality of M. hyopneumoniae genotypes from Brazilian farms affected by EP.

Identification of Brucella abortus biovar 4 of bovine origin in Colombia / Identificación de Brucella abortus biovar 4 de origen bovino en Colombia

The objective of this study was to identify twelve Brucella abortus isolates of bovine origin from the department of Nariño in Colombia up to the biovar level. These isolates are included in the collection of the Germplasm Bank of Microorganisms of Animal Health Interest -Bacteria and Virus (BGSA-BV). The identification was carried out through conventional methods such as macro and microscopic morphological descriptions, enzymatic activity, biochemical profile, substrate use and sensitivity to dyes. Complementary genotypic characterization was carried out using multiplex PCR for B. abortus, Brucella melitensis, Brucella ovis, and Brucella suis-Erytritol (AMOS-ERY-PCR), RFLP-IS711, by southern blot hybridization, as well as by the multiple locus variable number of tandem repeat analysis (MLVA) using the ery gene and the insertion sequence IS711 and variable number of tandem repeats (VNTR) as molecular markers. The results of the phenotypic and molecular characterization allowed to identify twelve isolates as B. abortus biovar 4 as well as to differentiate field from vaccine strains. This is the first study on the phenotypic and molecular identification of B. abortus isolates in Colombia. It was concluded that the phenotypic and molecular identification of twelve isolates as B. abortus biovar 4 could be achieved using conventional and molecular techniques with enough resolution power. The identification of these isolates to the biovar level in taxonomic and epidemiological terms will allow the use of this genetic resource as reference strains in future research. This finding constitutes the basis for identifying biotypes not previously reported in the country that might be useful to support brucellosis survey programs in Colombia.

El objetivo de este estudio fue identificar 12 aislamientos de Brucella abortus de origen bovino procedentes del departamento de Narino, Colombia, hasta la descripción de biovar. Estos aislamientos conforman la colección del Banco de Germoplasma de Microorganismos de Interés en Salud Animal, Bacterias y Virus. La identificación se hizo mediante métodos convencionales, como la descripción morfológica macro y microscópica de actividad enzimática, de perfiles bioquímicos, de utilización de sustratos y de sensibilidad a colorantes. Se hizo una caracterización genotipica complementaria mediante PCR múltiple para Brucella abortus, Brucella melitensis, Brucella ovisy Brucella suis-eritritol (AMOS-ERY-PCR); RFLP-/S7II; hibridación Southern blot y análisis multi-locus de repeticiones en tándem de número variable (MLVA), empleando como marcadores moleculares el gen ery, la secuencia de inserción /S711 y el número variable de repeticiones en tándem (VNTR). Los resultados de la caracterización fenotípica y molecular permitieron identificar 12 aislamientos de campo como B. abortus biovar 4 y diferenciar cepas de campo de cepas vacunales. Este es el primer estudio de identificación fenotípica y molecular de aislamientos de B. abortus en Colombia. Por su importancia taxonómica y epidemiológica, la identificación de estos aislamientos hasta el nivel de biovar permitirá disponer de recursos genéticos que se pueden emplear como cepas de referencia en futuras investigaciones. Estos resultados pueden considerarse como una base para la identificación de biotipos no reportados en el país y podrán ser utilizados en programas de monitoreo y vigilancia de la brucelosis bovina en Colombia.

The population structure of Ochrobactrum isolated from entomopathogenic nematodes indicates interactions with the symbiotic system.

Entomopathogenic nematodes (EPNs) form specific mutualistic associations with bioluminescent enterobacteria. In Heterorhabditidis indica, Ochrobactrum spp. was identified beside the symbiont Photorhabdus luminescens but its involvement in the symbiotic association in the EPNs remains unclear. This study describe the population structure and the diversity in Ochrobactrum natural populations isolated from EPNs in the Caribbean basin in order to question the existence of EPN-specialized clones and to gain a better insight into Ochrobactrum-EPNs relationships. EPN-associated Ochrobactrum and Photorhabdus strains were characterized by multi-locus sequence typing, Pulsed-Field Gel Electrophoresis fingerprinting and phenotypic traits. Population study showed the absence of EPN-specialized clones in O. intermedium and O. anthropi but suggested the success of some particular lineages. A low level of genetic and genomic diversification of Ochrobactrum isolated from the natural population of Caribbean nematodes was observed comparatively to the diversity of human-associated Ochrobactrum strains. Correspondences between Ochrobactrum and P. luminescens PFGE clusters have been observed, particularly in the case of nematodes from Dominican Republic and Puerto Rico. O. intermedium and O. anthropi associated to EPNs formed less biofilm than human-associated strains. These results evoke interactions between Ochrobactrum and the EPN symbiotic system rather than transient contamination. The main hypothesis to investigate is a toxic/antitoxic relationship because of the ability of Ochrobactrum to resist to antimicrobial and toxic compounds produced by Photorhabdus.