Molecular typing of canine distemper virus strains reveals the presence of a new genetic variant in South America

Canine distemper virus (CDV, Paramyxoviridae, Morbillivirus) is the causative agent of a severe infectious disease affecting terrestrial and marine carnivores worldwide. Phylogenetic relationships and the genetic variability of the hemagglutinin (H) protein and the fusion protein signal-peptide (Fsp) allow for the classification of field strains into genetic lineages. Currently, there are nine CDV lineages worldwide, two of them co-circulating in South America. Using the Fsp-coding region, we analyzed the genetic variability of strains from Uruguay, Brazil, and Ecuador, and compared them with those described previously in South America and other geographical areas. The results revealed that the Brazilian and Uruguayan strains belong to the already described South America lineage (EU1/SA1), whereas the Ecuadorian strains cluster in a new clade, here named South America 3, which may represent the third CDV lineage described in South America.     

Nodulation-gene-inducing flavonoids increase overall production of autoinducers and expression of N-acyl homoserine lactone synthesis genes in rhizobia

Legume-nodulating rhizobia use N-acyl homoserine lactones (AHLs) to regulate several physiological traits related to the symbiotic plant–microbe interaction. In this work, we show that Sinorhizobium fredii SMH12, Rhizobium etli ISP42 and Rhizobium sullae IS123, three rhizobial strains with different nodulation ranges, produced a similar pattern of AHL molecules, sharing, in all cases, production of N-octanoyl homoserine lactone and its 3-oxo and/or 3-hydroxy derivatives. Interestingly, production of AHLs was enhanced when these three rhizobia were grown in the presence of their respective nod-gene-inducing flavonoid, while a new molecule, C14-HSL, was produced by S. fredii SMH12 upon genistein induction. In addition, expression of AHL synthesis genes traI from S. fredii SMH12 and cinI and raiI from R. etli ISP42 increased when induced with flavonoids, as demonstrated by qRT-PCR analysis.     

Genomic analysis of pandemic and post-pandemic influenza A pH1N1 viruses isolated in Rio Grande do Sul,Brazil

During the 2009 influenza A pH1N1 pandemics in Brazil, the state that was most affected was Rio Grande do Sul (RS), with over 3,000 confirmed cases, including 298 deaths. While no cases were confirmed in 2010, 103 infections with 14 deaths by pH1N1 were reported in 2011. Genomic analysis of the circulating viruses is fundamental for understanding viral evolution and supporting vaccine development against these pathogens. This study investigated whole genomes of six pH1N1 virus isolates from pandemic and post-pandemic periods in RS, Brazil. Phylogenetic analysis using the concatenated genome segments demonstrated that at least two lineages of the virus co-circulated in RS during the 2009 pandemic period. Moreover, our analysis showed that the post-pandemic pH1N1 virus from 2011 constitutes a distinct clade whose ancestor belongs to clade 7. All six isolates contained amino acid substitutions in their proteins when compared to the archetype strains California/04/2009 and California/07/2009. The 2011 isolates contained more amino acid substitutions, and most of their genes were under purifying selection. Based on the amino acid substitutions in HA epitopes from strains isolated in RS, Brazil, in silico analysis predicted a decrease in vaccine efficacy against post-pandemic strains (median 31.562 %) in relation to pandemic ones (median 39.735 %).     

Phylogeographic dispersion and diversification of rabies virus lineages associated with dogs and crab-eating foxes (Cerdocyon thous) in Brazil

Genetic lineages of dog-associated RABV still circulate in some areas of the North and Northeast of Brazil. In parallel, another RABV lineage circulates among wild canids in the Northeast, particularly the crab-eating fox (Cerdocyon thous). Although previous studies and phylogenetic analyses have been carried out, the way in which these lineages are dispersed temporally and spatially remained to be elucidated. In this study, RABV N gene sequences isolated from canids in North and Northeast Brazil were analyzed by the Bayesian Markov Chain Monte Carlo Method, and the results were then used in a phylogeographic study. It was inferred from the findings that the most recent common ancestor became established at the end of the nineteenth century on the border of the Brazilian states of Paraíba and Pernambuco and diversified into the lineages associated with dogs and C. thous. Around 1910, the original C. thous lineage diversified into two main sublineages in the same area, one of which migrated to the south and the other to the north. The dog-associated lineage diversified around 1945 and moved toward the north and south. From the phylogeographic analysis it was possible to infer not only the movement of the virus lineages but also the probable location where dispersion and diversification occurred. The methodology used here enabled the phylogeographic history of RABV in the region to be reconstructed, and the dispersion pattern of the virus can be used to predict its movements, making it easier to stop the advance of a rabies epidemic.     

Extraintestinal pathogenic Escherichia coli (ExPEC) of human and avian origin belonging to sequence type complex 95 (STC95) portray indistinguishable virulence features

Extraintestinal pathogenic Escherichia coli (ExPEC) strains of certain genetic lineages are frequently implicated in a wide range of diseases in humans and birds. ExPEC strains belonging to the phylogenetic lineage/sequence type complex 95 (STC95) are one such prominent lineage that is commonly isolated from extraintestinal infections such as systemic disease in poultry and urinary tract infections (UTIs), neonatal meningitis and sepsis in humans. Several epidemiological studies have indicated that ST95 strains obtained from such infections may share similar virulence genes and other genomic features. However, data on their ability to establish infections in vivo as deduced from the manifestation of similar virulence phenotypes remain elusive.     

Salt tolerance of Rhizobium species in broth cultures

Salt tolerance of five rhizobia strains was examined in broth cultures. Five levels of NaCl concentration were used and the optical density was taken as a measure for the vigour of bacterial growth. Rhizobium leguminosarum and R. meliloti were tolerant to high levels of salinity and growth curves in saline broth showed a similar pattern to the control level. Rhizobium japonicum, cowpea Rhizobium, and R. trifolii were intolerant to salt and showed a strong growth retardation with increasing salt concentration. Growth was inhibited at high levels of salinity. It is suggested that rhizobia sensitivity to salts may be partly responsible to the inhibition of nitrogen fixation by legumes growing under salt stress.     

Mycobacterium tuberculosis Lineage 7 Strains Are Associated with Prolonged Patient Delay in Seeking Treatment for Pulmonary Tuberculosis in Amhara Region,Ethiopia

Recent genotyping studies of Mycobacterium tuberculosis in Ethiopia have reported the identification of a new phylogenetically distinct M. tuberculosis lineage, lineage 7. We therefore investigated the genetic diversity and association of specific M. tuberculosis lineages with sociodemographic and clinical parameters among pulmonary TB patients in the Amhara Region, Ethiopia. DNA was isolated from M. tuberculosis-positive sputum specimens (n = 240) and analyzed by PCR and 24-locus mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) analysis and spoligotyping. Bioinformatic analysis assigned the M. tuberculosis genotypes to global lineages, and associations between patient characteristics and genotype were evaluated using logistic regression analysis. The study revealed a high diversity of modern and premodern M. tuberculosis lineages, among which approximately 25% were not previously reported. Among the M. tuberculosis strains (n = 138) assigned to seven subgroups, the largest cluster belonged to the lineage Central Asian (CAS) (n = 60; 26.0%), the second largest to lineage 7 (n = 36; 15.6%), and the third largest to the lineage Haarlem (n = 35; 15.2%). Four sublineages were new in the MIRU-VNTRplus database, designated NW-ETH3, NW-ETH1, NW-ETH2, and NW-ETH4, which included 24 (10.4%), 18 (7.8%), 8 (3.5%), and 5 (2.2%) isolates, respectively. Notably, patient delay in seeking treatment was significantly longer among patients infected with lineage 7 strains (Mann-Whitney test, P < 0.008) than in patients infected with CAS strains (adjusted odds ratio [AOR], 4.7; 95% confidence interval [CI], 1.6 to 13.5). Lineage 7 strains also grew more slowly than other M. tuberculosis strains. Cases of Haarlem (OR, 2.8; 95% CI, 1.2 to 6.6) and NW-ETH3 (OR, 2.8; 95% CI, 1.0 to 7.3) infection appeared in defined clusters. Intensified active case finding and contact tracing activities in the study region are needed to expedite diagnosis and treatment of TB.     

Description of Leucocytozoon quynzae sp. nov. (Haemosporida,Leucocytozoidae) from hummingbirds,with remarks on distribution and possible vectors of leucocytozoids in South America

We describe Leucocytozoon quynzae sp. nov. (Haemosporida, Leucocytozoidae), which is the first Leucocytozoon parasite identified to species level in hummingbirds. It was found in the Amethyst-throated Sunangel (Heliangelus amethysticollis, Trochilidae, Apodiformes) captured in the Palacio Forest, which belongs to the damping zone of Chingaza National Natural Park, Cundinamarca, Colombia, at 2,900 m above sea level where the transmission occurs; the new species were found both in the high Andean forest and Paramo ecosystem. This parasite is described based on the morphology of its blood stages, a fragment of the mitochondrial cytochrome b gene, and the complete mitochondrial genome. Illustrations of blood stages of the new species are given, and the phylogenetic analysis places this lineage in a well-supported clade with other lineages of unidentified to species level leucocytozoids reported in the Trochilidae birds elsewhere. The new species possess gametocytes in roundish host cells; it can be readily distinguished from other similar leucocytozoids, primarily due to (1) a comma-like shape of the host cell nucleus, which extended one half or less of the circumference of the gametocyte and (2) a large number of prominent volutin granules in the cytoplasm. Identical mitochondrial cytochrome b sequence of Leucocytozoon quynzae was found in different hummingbird species at the type locality and also was reported in one passerine bird at the highlands of Peru. Leucocytozoon quynzae is the first leucocytozoid parasite described from South American birds; its transmission occurs both at low temperatures and high elevations. We discuss some patterns of distribution of avian leucocytozoids in South America and the role of Gigantodax spp. (Diptera, Simuliidae) as potential vectors of Leucocytozoon parasites in the Andean Region.     

Multilocus sequence analysis of Brazilian Rhizobium microsymbionts of common bean (Phaseolus vulgaris L.) reveals unexpected taxonomic diversity

The diazotrophic bacteria collectively known as “rhizobia” are important for establishing symbiotic N₂-fixing associations with many legumes. These microbes have been used for over a century as an environmentally beneficial and cost-effective means of ensuring acceptable yields of agricultural legumes. The most widely used phylogenetic marker for identification and classification of rhizobia has been the 16S rRNA gene; however, this marker fails to discriminate some closely related species. In this study, we established the first multilocus sequence analysis (MLSA) scheme for the identification and classification of rhizobial microsymbionts of common bean (Phaseolus vulgaris L.). We analyzed 12 Brazilian strains representative of a collection of over 850 isolates in addition to type and reference rhizobial strains, by sequencing recA, dnaK, gltA, glnII and rpoA genes. Gene sequence similarities among the five type/reference Rhizobium strains which are symbionts of common bean ranged from 95 to 100% for 16S rRNA, and from 83 to 99% for the other five genes. Rhizobial species described as symbionts of common bean also formed separate groups upon analysis of single and concatenated gene sequences, and clusters formed in each tree were in good mutual agreement. The five additional loci may thus be considered useful markers of the genus Rhizobium; in addition, MLSA also revealed broad genetic diversity among strains classified as Rhizobium tropici, providing evidence of new species.     

Markers of pathogenicity islands in strains of Aeromonas species of clinical and environmental origin

The aim of this study was to investigate the presence of markers of pathogenicity islands that may be informative to detect the virulent PAI carriers of clinical and environmental strains of Aeromonas spp. isolated in Mexico. virB2, virB9 and virB11 genes were found in Aeromonas strains isolated from environmental and clinical sources while cagE and tfc16 genes were only in strains of environmental origin. Having performed the wide screening presented in this study, we now have a set of strains to map and confirm the presence of a pathogenicity island in Aeromonas strains isolated in Mexico.     

Genomic variation and evolution of Staphylococcus aureus

The evolution of new human and animal pathogenic strains of Staphylococcus aureus has been due to the accumulation of mobile genetic elements (MGE) encoding methicillin resistance and virulence factors into successful lineages. These include epidemic methicillin-resistant S. aureus in hospitals (EMRSA), community-associated MRSA (CA-MRSA), fully vancomycin-resistant MRSA (VRSA) and livestock-associated MRSA (LA-MRSA). The S. aureus population in humans is dominated by about ten S. aureus lineages while animals generally have different lineages. Individual isolates within each lineage have unique combination of MGE often encoding virulence and resistance genes. S. aureus evolves due to point mutation and selection, but also dramatically due to the horizontal transfer of these MGE between strains or from other species or genera. Horizontal transfer, by conjugation or transduction, can be blocked by S. aureus restriction modification systems which are lineage specific. Because of the mobility of MGE, there are prospects for increasingly virulent and resistant strains to emerge that could severely affect healthcare and agriculture more effectively than the current pathogens.     

Haemosporidian parasites of a European passerine wintering in South Asia: diversity, mixed infections and effect on host condition

We studied haemosporidian parasites in the scarlet rosefinch Carpodacus erythrinus in a small isolated semicolony during an eight-year period using molecular methods of parasite detection. The scarlet rosefinch is an interesting model of parasite host species. It winters in South Asia which represents a rare exception among European passerines. Males express yellow to red carotenoid-based plumage ornament which is a good predictor of male reproductive success. In 240 blood samples originating from 199 adult individuals, the total parasite prevalence reached 60 %. Prevalence varied among years from 36 to 81 % in Haemoproteus, 8 to 22 % in Plasmodium, and 0 to 14 % in Leucocytozoon. Twenty parasite lineages were detected (Haemoproteus: 5 lineages, Plasmodium: 10 lineages, and Leucocytozoon: 5 lineages). Among them, the Haemoproteus ROFI2 lineage, which is a host-specific parasite lineage of the scarlet rosefinch, was the most frequently found. Parasite lineages showed varying degree of lineage specificity. While Haemoproteus lineages detected in the scarlet rosefinch have relatively narrow host breadth restricted mainly to Fringillidae family, Leucocytozoon and Plasmodium lineages generally showed wider host range. The presence of some parasite lineages hitherto detected in sedentary European passerines (SISKIN1, CCF3, BT2) or in Culicoides biting midges at the same locality (ROFI1) suggest local transmission. On the contrary, lineages LK05 and FANTAIL1 that were previously reported exclusively from Asian hosts imply parasite transmission at the scarlet rosefinch wintering sites in South Asia. Mixed infections were found in 17 % of infected samples and comprised mainly the most frequent lineages. The pattern of concomitant infections seemed to be rather random and matched expected levels based on lineage frequencies. Between-year comparisons revealed that in a majority of the repeatedly captured individual hosts the infection status remained unchanged (individuals stayed uninfected or possessed the same parasite lineages). However, 16 gains and 8 losses of lineages were also reported. We have not found any effect of haemosporidians on male carotenoid ornament expression or host body mass.     

Genomic characterization of echovirus 6 causing aseptic meningitis in Hokkaido, Japan: a novel cluster in the nonstructural protein coding region of human enterovirus B

We determined four complete nucleotide sequences of echovirus 6 (E6) isolated from an epidemic of aseptic meningitis (AM) in Hokkaido, Japan, in 2011. Phylogenetic analysis of the genes encoding viral capsid protein 1 revealed that the strains were closely related to E6 strains isolated in China in recent years, but they were distantly related to E6 strains isolated from patients with AM in Osaka Prefecture, Japan, in 2011. The genes encoding the viral protease and RNA-dependent RNA polymerase (3CD) were closely related to those of several non-E6 strains of the species Human enterovirus B isolated in China, South Korea, and Australia from 1999 to 2010, resulting in a novel cluster in the phylogenetic tree. These results suggest that the incidence of AM in Japan in 2011 was caused by at least two lineages of E6 strains, and a lineage of the 3CD gene was interspersed among different serotypic strains isolated in Western Pacific countries.     

Characterization of two new rhabdoviruses isolated from midges (Culicoides SPP) in the Brazilian Amazon: proposed members of a new genus, Bracorhabdovirus

Summary. Itacaiunas and Curionopolis viruses were isolated from Culicoides midges in Parauapebas municipality, Pará state, Brazil, in 1984 and 1985, respectively. Itacaiunas virus infected newborn mice and mosquito cells (C6/36), but did not replicate in some mammalian cell lineages; while Curionopolis virus infected only mice. Neither virus showed a serological relationship with any of the 195 known arboviruses circulating in Brazil, nor against 38 other rhabdoviruses isolated worldwide. Both virus particles are bullet-shaped and similar in morphology to that observed for other members of the family Rhabdoviridae. Partial nucleotide sequencing of the N protein showed that those two viruses constitute a separate clade in the family Rhabdoviridae, which we propose to be a new genus, designated Bracorhabdovirus.     

Changes in the Population of Methicillin-Resistant Staphylococcus pseudintermedius and Dissemination of Antimicrobial-Resistant Phenotypes in the Netherlands

Methicillin-resistant Staphylococcus pseudintermedius (MRSP), which is often multidrug resistant (MDR), has recently emerged as a threat to canine health worldwide. Knowledge of the temporal distribution of specific MRSP lineages, their antimicrobial resistance phenotypes, and their association with clinical conditions may help us to understand the emergence and spread of MRSP in dogs. The aim of this study was to determine the yearly proportions of MRSP lineages and their antimicrobial-resistant phenotypes in the Netherlands and to examine possible associations with clinical conditions. MRSP was first isolated from a canine specimen submitted for diagnostics to the Faculty of Veterinary Medicine of Utrecht University in 2004. The annual cumulative incidence of MRSP among S. pseudintermedius increased from 0.9% in 2004 to 7% in 2013. MRSP was significantly associated with pyoderma and, to a lesser extent, with wound infections and otitis externa. Multilocus sequence typing (MLST) of 478 MRSP isolates yielded 39 sequence types (ST) belonging to 4 clonal complexes (CC) and 15 singletons. CC71 was the dominant lineage that emerged since 2004, and CC258, CC45, and several unlinked isolates became more frequent during the following years. All but two strains conferred an MDR phenotype, but strains belonging to CC258 or singletons were less resistant. In conclusion, our study showed that MDR CC71 emerged as the dominant lineage from 2004 and onward and that less-resistant lineages were partly replacing CC71.     

Genetic Identification of Rickettsial Isolates from Fatal Cases of Brazilian Spotted Fever and Comparison with Rickettsia rickettsii Isolates from the American Continents

Fifteen bacterial isolates from spotted fever group rickettsiosis in Brazil were genetically identified as Rickettsia rickettsii. In a phylogenetic analysis with other R. rickettsii isolates from GenBank, the Central/South American isolates showed low polymorphism and formed a clade distinct from two North American clades, with the North American clades having greater in-branch polymorphism.     

Brazilian P[8],G1, P[8],G5, P[8],G9, and P[4],G2 rotavirus strains: nucleotide sequence and phylogenetic analysis

Rotavirus epidemiological surveys with molecular analysis of strains are required for gastroenteritis control and prevention. Twenty-nine human rotavirus strains detected in Rio de Janeiro, Brazil, from 1986 to 2004 were characterized as P[8],G1, P[8],G5, P[8],G9, and P[4],G2 genotypes. The VP7 genes were sequenced and phylogenetic analysis was performed. Strains of genotype G1 revealed two distinct lineages, G1-3 and G1-4; strains of genotype G2 grouped in lineage G2-1; G5 strains clustered with other Brazilians G5 strains and G9 strains were closely related to each other in lineage G9-3, distinct from the original G9 strains detected in 1980s. The VP4 genes were analyzed and P[8] strains fell into two major genetic lineages, P[8]-2 and P[8]-3. Our findings document an intragenotype diversity represented by lineages and sublineages within rotavirus circulating in Rio de Janeiro from 1986 to 2004, before application of a vaccine (Rotarix) in Brazil. This report emphasizes the importance of continuing monitor genotypes to verify if uncommon strains or newly strains are emerging to be specifically addressed in future vaccine trials.     

Phylogenetic Placement of Rickettsiae from the Ticks Amblyomma americanum and Ixodes scapularis

A rickettsial isolate (isolate MOAa) belonging to the spotted fever group (SFG) was obtained from the lone star tick Amblyomma americanum. We used PCR to characterize the genes for the rickettsial outer membrane proteins rOmpA and rOmpB. We sequenced the PCR products (domains I of both the rompA gene and the rompB gene) of MOAa and WB-8-2, another rickettsial isolate from A. americanum. To place MOAa and WB-8-2 and two other nonpathogenic isolates (Rickettsia rickettsii Hlp2 and Rickettsia montana M5/6) with respect to their putative sister species, we included them in a phylogenetic analysis of 9 Rickettsia species and 10 Rickettsia strains. Our phylogenetic results implied three evolutionary lineages of SFG rickettsiae and that WB-8-2 and MOAa were most closely related to R. montana. New World isolates were not the most closely related to each other (they did not form a clade). Rather, our results supported four independent origins (introductions) of rickettsiae into North America from different Old World regions. The results of our phylogenetic analysis did not support the hypothesis of a stable coevolution of rickettsiae and their tick hosts. Finally, we examined the rompA gene of a nonpathogenic rickettsial symbiont isolated from the tick Ixodes scapularis. In a phylogenetic analysis, the symbiont was placed as the sister to R. montana and its isolates. The relationship of this symbiont to R. montana raised questions as to the potential origin of pathogenic SFG rickettsiae from nonpathogenic tick symbionts, or vice versa.     

Population structure and genetic diversity of Actinobacillus actinomycetemcomitans strains isolated from localized juvenile periodontitis patients

The phylogeny of 20 Actinobacillus actinomycetemcomitans strains isolated from patients with localized juvenile periodontitis (LJP) was investigated by using partial sequence analysis of 16S rRNA genes, arbitrarily primed PCR (AP-PCR), and four additional PCR assays that amplified polymorphic regions in the leukotoxin (lkt), cytolethal distending toxin (cdt), major fimbrial subunit (flp-1), and serotype-specific O polysaccharide gene clusters. Our analysis also included four strains isolated from healthy subjects and nine reference strains. We found that A. actinomycetemcomitans strains comprised three major phylogenetic lineages. One lineage consisted of serotype b strains, a second lineage consisted of serotype c strains, and a third lineage consisted of serotype a, d, e, and f strains. 16S rRNA sequences within each lineage were highly conserved (<1% base substitutions), whereas sequences between lineages were exceptionally divergent (1.9 to 5.0% substitutions). Two strains exhibited 16S rRNA sequences that were even more distantly related to those of the three major lineages (2.7 to 6.7% substitutions), indicating that additional minor lineages or variants exist. The distribution of 16S rRNA sequences and lkt, cdt, flp-1, and AP-PCR genotypes was consistent with a clonal population structure, with little evidence of assortative recombination between strains of different serotypes. Strains from all three major lineages were recovered from LJP patients, suggesting that phylogenetically diverse strains of A. actinomycetemcomitans carry pathogenic potential.     

Phylogenetic analysis of human P[8]G9 rotavirus strains circulating in Brazil reveals the presence of a novel genetic variant

BackgroundGroup A rotavirus (RV-A) genotype P[8]G9 has emerged as one of the leading causes of gastroenteritis in children worldwide and currently is recognized as one of the five most common genotypes detected in humans. High intragenotype diversity in G9 RV-A has been observed, and nowadays, based on the genetic variability of the VP7 gene, six different phylogenetic lineages and eleven sublineages were described.ObjectivesTo study the degree of genetic variation and evolution of Brazilian P[8]G9 RV-A strains.Study designPhylogenetic analysis of 19 P[8]G9 RV-A strains isolated from 2004 to 2007 in five different Brazilian states was conducted using the NSP1, NSP3, NSP5, VP4 and VP7 genes. For the VP4 and VP7 genes, 3D protein structure predictions were generated to analyze the spatial distribution of amino acid substitutions observed in Brazilian strains.ResultsBased on the phylogenetic analyses, all Brazilian strains clustered within lineage G9-III and P[8]-3 for VP7 and VP4, respectively, and were classified as genotype A1, T1 and H1 for the NSP1, NSP3 and NSP5 genes, respectively. Interestingly, all the strains isolated in Acre State (Northern Brazil) formed a closely related cluster clearly separated from the other Brazilian and prototype strains with regard to the five genes studied. Unique amino acid substitutions were observed in Acre strains in comparison with the prototype and Brazilian strains.ConclusionInclusion of Acre strains in the phylogenetic analysis revealed the presence of a novel genetic variant and demonstrated a diversification of P[8]G9 rotaviruses in Brazil.