AbstractEupsophus (ground frogs) inhabits exclusively the temperate forests of southern South America (Chile and Argentina). The current delimitation of the genus was reached in the late 1970s, when only two species were recognized, but since then the number of described species steadily increased, reaching a maximum of 11 by 2012. Subsequent studies that applied explicit species delimitation approaches decreased the number of species to six in 2017 and raised it again to 11 the following year, including an undescribed putative species. Despite these taxonomic changes, the two species groups traditionally recognized, roseus and vertebralis, have been maintained. Another recent contribution to the taxonomy of the genus was the explicit recognition of the extremely high level of external phenotypic variation exhibited by species of the roseus group, which undermines the utility of some diagnostic characters. Here we provide a critical review of the extensive taxonomic and systematic literature on the genus over the last six decades, to examine the evidence behind the recurrent taxonomic changes and advances in its systematics. We also update and complete a 2017 review of geographic information, provide additional qualitative observations of external characters commonly used in the diagnoses of species of the roseus group, and reassess the phylogenetic position of a putative new species from Tolhuaca (Chile), which was not included in the last species delimitation study. The present review shows that: 1) there is no congruence between the patterns of phenotypic and genetic/phylogenetic differentiation among species of both groups; 2) in the roseus group, the intraspecific variation in some external characters is as high as the differences described among species; 3) there is little morphological and bioacoustic differentiation within species groups, and inconsistencies in the chromosomal evidence at the genus level; 4) under the latest taxonomic proposal (2018), species of the roseus group still lack consistent and reliable diagnoses and their distribution limits are poorly defined; and 5) the population from Tolhuaca represents an additional undescribed species under the most recent taxonomic framework. Finally, we discuss the implications of these findings for the taxonomy and biogeography of the genus, pointing out some areas that require further research to understand their patterns and processes of diversification.The genus  Alsodidae, which currently is represented there by two sister genera, Alsodes Bell, 1843  is found exclusively in temperate forests, inhabiting mainly the forest floor , E.calcaratus , E.insularis , E.migueli Formas, 1978, E.contulmoensis Ortiz, Ibarra-Vidal & Formas, 1989, E.nahuelbutensis Ortiz & Ibarra-Vidal, 1992, E.septentrionalis Ibarra-Vidal, Ortiz & Torres-Pérez, 2004, E.altor Nuñez, Rabanal & Formas, 2012, and a putative new species from Villarrica, Chile) and vertebralis  (Temperate forests of southern South America (Chile and Argentina) are home to a reduced but evolutionarily diverse group of amphibians . The mosst floor . Recentlterature , accorditerature  are arras, 1989) .Eupsophus has a long and complex taxonomic history. Among the currently valid species, the first two were described in the nineteenth century under other genera: Cystignathusroseus and Cacotuscalcaratus  insularis is currently recognized. The use of the name Eupsophus, coined by Alsodes, Batrachyla, Phrynopus, Thoropa; e.g., E.roseus and E.vertebralis). Since 1978, when E.migueli was described , but one of them, E.queulensis, was synonymized with E.septentrionalis by The genus escribed , the numroseus group. Two of these studies . E.queulensiswith E.septentrionalis and suggested that the population from Tolhuaca, also located in the Chilean Andean foothills (38°13'S), would correspond to an undescribed species related to E.roseus. More recently, During the last six decades, morphometric, immunological, chromosomal, bioacoustic and molecular  approaches have been applied, separately or in combination, to the taxonomy and systematics of these frogs  in this group. On the other hand, the taxonomic proposal by The application of molecular approaches and integrative taxonomy to the discovery and delimitation of species has drastically changed our estimates of amphibian diversity at global and local levels . Recent ed above , the des applied , and the applied , based oaracters  and moleEupsophus was roseus group , information about the genus, in its current definition  are Eupsophus and was the most comprehensive molecular phylogenetic study of the genus until E.roseus as Cystignathusroseus), E.calcaratus as Cacotuscalcaratus), E.insularis as Borborocoetus (Cystignathus) insularis), E.vertebralis and E.roseus, the latter as E.grayi), E.insularis as subspecies of E.grayi), E.migueli), E.calcaratus and E.insularis), E.emiliopugini), E.contulmoensis), E.nahuelbutensis), E.septentrionalis), E.queulensis, synonymized with E.septentrionalis by E.altor). Other studies of Eupsophus with a taxonomic and/or systematic focus have used different approaches: Our literature review was focused on (but not restricted to) taxonomic, genetic and phylogenetic studies in which phenotypic and/or genetic variation within and among on e.g., , is easiWe compiled literature records to define the geographic ranges of the 11 species recognized by roseus group  vary at the intrapopulation level. Here, we provide additional examples of intrapopulation variation in the first three characters. The observations were made in two undescribed and two type localities , including less than 20 live specimens per locality. All specimens were released at the same capture site after being photographed.E.roseus. E.roseus. th generation. The first 25% of generations were conservatively discarded as burn-in after observing the stationarity of ln-likelihoods of trees in Tracer v1.7 .cer v1.7 . ConvergEupsophus, because they combined several invalid  into two taxa, which represent the two species groups currently recognized , most of them derived from or closely related to E.roseus. One additional species from Isla Wellington (southern Chile), closely related to E.calcaratus, was proposed by E.calcaratus). All descriptions and revalidations  were primarily motivated by observations of differences in external morphological characters and in some cases also internal ones. Other types of characters were added in some descriptions and diagnoses . They extracted the information mainly from the original diagnoses, but also used other two sources for E.roseus, E.calcaratus and E.insularis, since the original descriptions and diagnoses of these species are very brief and were made under generic names no longer used. The two additional sources are E.calcaratus and E.insularisare revalidated, and roseus group: 1) in some cases, characters that varied in the type series were used; 2) the great heterogeneity in number and type of characters used, which makes it difficult to identify the differences among the species; and 3) the four characters most frequently included in the diagnoses vary widely at the intraspecific level. Here  or these could be differentiated only by the body coloration pattern , which has been described as variable in most species  from being later included as a diagnostic character for several species of the group . There are also previous literature records of variation in the other three characters mentioned. The coloration of the iris has been included recurrently in the descriptions and diagnoses of the species of the roseus group, so it was considered a useful character to distinguish certain species . The shape of the distal end of the xiphisternum is the osteological character most frequently included in descriptions and diagnoses , but in E.insularis it is truncated and slightly notched (E.migueli it is notched (E.emiliopugini). However, one study  and of E.migueli , finding four types of xiphisternum  in E.migueli and three in E.roseus (notched condition was not found). Although in both species the rounded xiphisternum was the most frequent condition, this example demonstrates that intrapopulational variation in osteological characters may be detected when a large number of specimens is examined. up Table . Moreovelcaratus , E.emiliiopugini  and E.alis group , but als.migueli , E.calcalcaratus , E.contulmoensis , E.nahuebutensis  and E.serionalis . Correa es Table . In contellowish . Iris cops Table . For exae former  and shore former ; Table 1 notched   and the other species  that the genus contained at that time. E.grayi , but in those groups of populations he mixed several species that were described later. Subsequent studies on adults have applied multivariate statistical techniques , but they have been carried out with a small number of species  and populations (no study included more than one population per species). Despite these limitations, morphometric differences have been observed between the species groups , but scarce differences within them.Morphometric approaches have usually been used to infer, implicitly or explicitly, the relationships among species or to discriminate  them. Also, they have been used in conjunction with allozymes (see below) to evaluate explicitly the agreement between morphological and genetic evolution in the genus . The firs groups , and nots groups . In partEupsophus currently recognized are shown in Table E.nahuelbutensis has not been described, although roseus group, 28 in the vertebralis group; E.migueli, E.insularis, E.septentrionalis, E.roseus the sex chromosomes do not differ in form, but can be distinguished by their constitutive heterochromatin patterns  or several localities  can be compared. Almost all karyotypes of the same species described by different authors differ in chromosomal morphology and position of the secondary constriction, and even in the presence or absence of this last structure , so that intrapopulation and/or intraspecific variation is revealed only when different studies are compared. The levels of variation in chromosome morphology and position of the secondary constriction within a same species  are as high as the levels of variation observed among species of the same group , and less differentiation between E.septentrionalis and its geographically closest congeners, E.contulmoensis and E.nahuelbutensis, were reported. These allozyme patterns, particularly the almost fixed differences between E.septentrionalis and E.roseus, were used to support the specific status of E.septentrionalis  using two restriction enzymes.Since the mid-1970s, several immunological techniques and enzymatic systems  were used to solve taxonomic and systematic problems of the anurans of the temperate forests of South America, including the genus  enzymes , 1986 has groups  than wits groups . In fact.migueli , and E.cbutensis  are almorionalis ; Table 1omosomal  and bioaomosomal  evidenceomosomal  used thiStudies with DNA sequencesEupsophus, its phylogenetic position with respect to other anuran groups, the phylogeographic history of one of its species  and its species diversity with species delimitation approaches  as “diagnostic of species lineages” . E.altor, performed a phylogenetic analysis with a fragment of the control region , in which a sister relationship between E.altor  and E.migueli was recovered , but an examination of the sequences of E.altor; see comment in Eupsophus and Alsodes with mitochondrial and nuclear genes, including the 11 nominal species of Eupsophus recognized at that time, and in some cases more than one locality per species . E.roseus , and estimated the phylogenetic relationships within the genus, using mitochondrial and nuclear sequences and including a greater number of specimens and localities than roseus group consistent with that of E.calcaratus in a different position with respect to previous studies . They also obtained a weak support for an alternative position of E.septentrionalis, which is congruent with previous hypothesis  and E.contulmoensis + E.nahuelbutensis, the position of E.calcaratus as sister taxon of all the other species of the roseus group), with the notable exception of E.septentrionalis, whose position fluctuates between studies  is uncertain, since both have not been included simultaneously in any study  had distributions surrounded by populations of other species according to literature records. On the other hand, E.calcaratus). Although roseus group have restricted distributions, maintaining the same pattern of overlap between some species that is derived from the accumulated information of the literature    marks thopugini) .roseus group is the same as that of the genus  generated with point occurrences and areas, respectively. roseus group and the Chilean portion of the distribution. However, their maps (their fig. 2) were only intended to represent the boundaries among species that can be inferred by combining all the occurrence points collected from the literature. roseus group exhibited a high degree of overlap, including several cases of the presence of more than one species in the same locality reported in the same or different publications .One of the six species of the Taxon classificationAnimaliaAnuraCycloramphidaeValdivia , b; locaE.migueli, E.contulmoensis, E.nahuelbutensis and E.altor), without clarifying the level of sympatry between them. In fact, E.roseus has been recorded in the type localities of some of these species: M.N. Contulmo , and includes the few confirmed localities from Argentina (see below). Also, this map covers completely the distribution ranges of E.migueli and E.altor, and the continental area where E.insularis has been recorded (see below). According to E.roseus in Chile would be the Calle-Calle River basin (approximately 39°50'S), from where would be replaced by E.calcaratus southwards. The map of the E.roseus further south, until Cordillera Pelada  and demonstrated that effectively there are populations of E.calcaratus north of some localities of E.roseus. Taken together, these last two studies show that both species are present in Chile between 39°55' and 40°20'S approximately, although the degree of sympatry between them is currently unknown. Until 1996 , E.roseus was considered as the only species of the genus in Argentina  shows that this species reaches further south through the Chilean Andes, which suggests the need to reevaluate the taxonomic status of the populations located in Argentina at the same latitude.The distribution range of this species is the most difficult to define from the literature, because its distribution limits differ among sources and four species were described within its range in Chile , recently reported by E.roseus, but in this case these findings are supported by molecular evidence. Together with Naguilán  and the Osorno coast (40°49'S). Only two localities outside this area are known, Tolhuaca , on the western margin of the Andes, and Puerto Blest in Argentina 107; , on the Taxon classificationAnimaliaAnuraCycloramphidaeLa Picada ; localitEupsophusemiliopugini would be distributed both on the coast and the Andean zone, mainly in Chile, between 40°11' and 45°30'S, although it would be in sympatry with E.vertebralis in a small area of the Chilean Coastal Range (see above). In Argentina, it is present on the northwest and southwest coasts of Lago Puelo , but as E.roseus because they make up a well-supported monophyletic group with specimens from the type locality of that species . The two new localities where phenotypic observations were done for this review (see below) are also included here.The two undescribed species mentioned in the recent literature Fig.  are knowEupsophus species , no specimens with the iris orange like E.roseus were observed.One of the contributions of eli Fig.  were coleli Fig. . At Mehuroseus group as well-supported clades , but the topology within this group is variable among analyses, including some polytomies, and only partially congruent with previous phylogenetic studies , which was reduced to 998 when cutting ends with gaps . The four analyses  recovered the two species groups and all the currently recognized nominal species of the ies Fig. . Figure sis Fig.  is the pEupsophus with respect to its sister genus, Alsodes, is now well established based on morphological, chromosomal, bioacoustic, developmental and molecular phylogenetic evidence . These and additional examples from other populations show that variation in body coloration is widespread in the genus, but this phenomenon has rarely been recognized in the literature (E.insularis and E.migueli). These high levels of variation in diagnostic characters have deep consequences for the current taxonomy , suggesting strongly observer biases  described within of the distribution range of E.roseus, whose limits and degree of sympatry have been never precisely established. The records compiled here also show an overlap between the distribution ranges of E.roseus and E.calcaratus, which is partially supported by molecular evidence but does not coincide with the previously established limits  have “restricted distributions”, which implies that the genus would have a highly fragmented distribution at present. This pattern is incompatible with the information available since there are historical records of E.roseus , field identification would be reliable only within the assumed distribution ranges and, as we have demonstrated, there has not been consensus about them. Therefore, inconsistent diagnoses, field misidentifications and misleading geographic data might be intimately linked, explaining most cases of sympatry and range overlap inferred from the compilation of localities. In turn, erroneous geographic data might influence the identification of atypical specimens, particularly in the distribution limits and unexplored zones. The problem of field misidentification is expected to persist under the most recent taxonomic arrangement  and, as we pointed out above, the diagnoses of Eupsophus species are unreliable and their geographic boundaries are still poorly defined.This review summarizes six decades of taxonomy and systematic research on angement  since thEupsophus. However, except for roseus group  has important consequences for the biogeography and taxonomy of the genus. Historical records and intermediate undetermined populations show that this pattern of isolated species does not adequately reflect the distribution of the genus, but more importantly, some of these populations occupy intermediate phylogenetic positions between some narrow-range species of the roseus group, decreasing the genetic divergence among them (Eupsophus, whether molecular or not, should take into account the incongruities between the patterns of molecular, morphological, bioacoustic and chromosomal divergence and incorporate more intermediate populations to obtain a more accurate estimate of its species diversity.In this context, phylogenetic and species delimitation studies with DNA sequences have emerged as an independent and powerful way to reassess the taxonomy of ong them . The lat


Escherichia coli (E. coli) is one of the seven important types of microbial resistance. This meta-analysis study was conducted to identify the prevalence of cefepime-resistant E. coli in Iran during 2007–2016.Cefepime-resistant 2) between the reviewed studies. The collected data were categorized into different subgroups on the basis of the year of study and province. Data analysis was performed using the Statsdirect software.Studies published were searched in Persian databases  and international databases . The selected studies included all types of laboratory methods. Moreover, the random effects model (DerSimonian Laird method) was used to addresses the high heterogeneity , ranging from 25%  in 2009 to 61.95%  in 2016.Overall, 516 articles were selected from the searched medical databases. After reviewing and applying the inclusion criteria, irrelevant papers were excluded and the remaining 26 studies were meta-analyzed. The overall prevalence of cefepime-resistant E. coli in Iran has had an increasing and alarming trend during the recent years. Therefore, it is necessary to use practical strategies and interventions to control and monitor cefepime-resistant E. coli in the country.The prevalence of cefepime-resistant  E. coli is one of the most important germs. Escherichia coli is a gram negative bacteria, anaerobic, spore free, and normal flora that is the main cause of more than 80% of the acquired urinary tract infections  is one of the most important challenges facing the public health and is introduced by (WHO) as a growing major health threat. Given the increasing spread of AMR in the world, the WHO recommends controlling and monitoring AMR and the use of antibiotics , 2. Cefefections . Infectiountries . DiarrheE. coli transmission , studies performed on non-humane samples, and studies with duplicated data were also excluded.Exclusion criteria: Studies that did not estimate the prevalence of cefepime-resistant E. coli without especial antimicrobial susceptibility test type were included.Initially, the Persian and English databases were separately searched by two of the researchers. To select the related papers, their title, then their abstracts, and finally their full text were reviewed. At each stage, in case of controversy, consensus was achieved through consulting with the supervisor of the research team to make a final decision. The lists of references of the selected studies were also evaluated and the relevant articles were included in the study. All studies that estimated the prevalence of cefepime-resistant Data on authors’ name, date of publication, time of study (year), place and type of study, sample size, gender, mean age of samples, and type of antibiotic resistance were extracted from the selected cross-sectional studies performed on human samples. The collected data was entered into Statsdirect software for final analysis.Strobe checklist was used to assess publication bias and quality of the selected studies . Strobe After evaluating the aforementioned cases, the studies were divided into three groups: studies with high, moderate, and low bias. Low-bias and high-quality studies are studies that meet all the seven items listed in Strobe checklist. Studies with moderate bias and moderate quality are studies that meet six items of Strobe checklist. Studies with high bias and low quality are studies that do not meet two or more items of Strobe checklist. The criteria used to measure bias in the selected studies include: bias in outcome evaluation, bias in exposure assessment, sample size calculation, information bias, and selection bias. All stages of the quality assessment of the articles were also conducted by two researchers independently. PRISMA checklist (2009) was used to assess the quality of systematic review and meta-analysis .E. coli calculated as P (ratio) with a confidence interval (CI) of 95%. To test heterogeneity, Q test was used at an error level of less than 10%, and its quantity was calculated using I2 index. I2 is an index used for estimating the variance between studies. Considering the results of heterogeneity test, random effects model was used for data analysis at a confidence interval of 95% (2) in the study, the data was analyzed using DerSimonian Laird method. Data analysis was also performed on subgroups there were determined based on the province of residence and year of study. Beg rank correlation test was used to measure publication bias. A P-value<0.1 indicates a significant bias. A funnel plot was used to evaluate the publication bias. In addition, a significance level of α=0.05 was used in bilateral statistical tests . The level of heterogeneity in the prevalence rates was 96.5%, which is consistent with the Cochrane definition in 2008 E. coli iE. coli was 61.95%  in 2016 and 25 %  in 2009. The heterogeneity among the studies  was calculated through the quantitative evaluation via Beg test= . Considering province of study, the prevalence of cefepime-resistant E. coli was estimated to be 70%  in Markazi and 15.32%  in Kerman and there was a heterogeneity between the studies   .E. coli. Beg test was used for quantitative evaluation of the publication bias .A funnel graph was used to show the distribution bias in studies conducted on the prevalence of cefepime-resistant E. coli at a confidence interval of 95%, each study was excluded and the results were compared with and without the results obtained through the analysis of all the studies. After the exclusion of each of the studies one by one, the results of susceptibility analysis showed that none of the studies alone had a significant effect on the overall prevalence . The lowest and highest sample sizes were observed in the provinces of Mazandaran and East Azerbaijan, respectively. From among the ten studied years, the highest prevalence rate was observed in 2016, indicating an increase in the resistance to antibiotics in recent decades. The highest and lowest prevalence rates, respectively, were 61.95%  in 2016 and 25%  in 2007.Twenty six studies had been conducted to determine the prevalence of cefepime-resistant 04 cases . The oveE. coli in Iran was inconstant and varied from 15.32% to 100%    . In Maza87, 100) . The lowE. coli was studied. In all provinces of Iran, no study had been performed on the prevalence of cefepime-resistant E. coli. However, an overall estimation in the country could be obtained from the prevalence rates reported in the nine studied provinces. In all the studied years, no study was performed on cefepime-resistant E. coli. In these studies, no special and unique laboratory method was applied. Moreover, in meta-analysis studies, there is always the possibility of losing some articles taken into account.This study had some limitations, for instance no specific species of E. coli in Iran is high and has an increasing trend. This increasing trend is a challenge to be resolved. It is necessary to adopt appropriate measures and interventions.The prevalence of cefepime-resistant Ethical issues  have been completely observed by the authors.


Sporothrix schenckii, a dimorphic organism that was first described more than a century ago in human and rat specimens [Sporothrix have been changing significantly, and sporotrichosis is now considered a zoonotic disease, caused not only by one fungal species, and is associated with high morbidity rates, and, in the immunocompromised host, mortality caused by this infection is usual [Sporothrix genus, but a handful of them, namely the pathogenic clade composed of S. schenckii, Sporothrix brasiliensis, Sporothrix globosa, and Sporothrix luriei, are related to sporotrichosis [Sporotrichosis is a neglected, deep-seated fungal infection traditionally associated with pecimens . Since tpecimens . Howeveris usual ,4,5. Thurichosis . This SpS. brasiliensis has only been found in Brazil, whereas S. globosa predominantly in Asia; S. schenckii, on the other hand, is distributed worldwide [S. schenckii is indeed the most prevalent species isolated from clinical specimens collected in Mexico, with the absence of S. brasiliensis and an anecdotic isolate of S. globosa [So far, orldwide . As a co globosa .Sporothix spp. [Since the first description of the disease, its diagnosis has relied on the clinical manifestations, the direct examination of biological specimens, and the culturing of the microorganism . Howeverhix spp. . The michix spp. . The culhix spp. . Moreovehix spp. . The stuhix spp. .Sporothix spp. has been traditionally related to vegetal tissues, debris, and decaying matter; this is the reason sporotrichosis is known as the rose pickers’ disease or the gardener’s disease [S. brasiliensis [The natural niche of  disease ,3,4. Mosiliensis . The curiliensis . One proiliensis . Here, diliensis .Sporothrix genus are poorly understood and limited information is currently available. Here, Ramírez-Soto et al. report a retrospective study aiming to establish the environmental conditions of the geographical locations where these organisms have been isolated from soil samples [Sporothrix was commonly isolated, and S. schenckii was the most frequent species retrieved from these samples with an estimated temperature ranging from 6.6 °C to 28.84 °C and a relative humidity between 37.5% and 99.06% [Sporothrix spp., Batista-Duharte et al. studied the effect of the exposure to mercury (II) chloride on the susceptibility to sporotrichosis [The specific environmental and ecological aspects of the natural habitat of the pathogenic species of the  samples . Sixteend 99.06% . To provrichosis . This harichosis . The merrichosis .Sporothrix spp., as discussed here by García-Carnero et al. [Sporothrix vaccine [Sporothrix response [S. schenckii cell wall proteins and then cocultured with splenocytes. As a result, the activated dendritic cells stimulated a cytokine profile suitable for the activation of the Th1 cell population, suggesting the cell wall preparation could lead to the establishment of a protective antifungal response [The antifungal repertoire to treat sporotrichosis usually depends on the clinical form of the disease, the longevity of the infection, the presence of other chronic illnesses in the patient, and the fungal species. Terbinafine, potassium iodine, and some azoles are among the drugs with good antifungal activity against o et al. . Due to  vaccine ,21,22,23 vaccine . Here, Qresponse . Mouse bresponse . Since tSporothrix spp. or any other pathogen requires basic knowledge of the host–pathogen interaction, the main determinants that assist the invader in colonizing and damaging tissues, as well as the antigenic molecules this may possess and which are therefore recognized by elements of the immune response. Here, Conceição-Silva and Morgado and García-Carnero et al. summarize the current knowledge on the recognition of Sporothrix by the host immune system and identify the challenges that should be addressed in the near future [Without doubt, the development of rational strategies to generate protective immunization against r future ,16.


Phlebotomus vector species with proven or suspected vector competence for five Leishmania pathogens occurring in Eurasia and Africa. A GIS-based spatial overlay analysis was then used to compare the distributional patterns of vectors and pathogens to help evaluate the vector species–pathogen relationship currently found in the literature. Based on this single factor of vector incrimination, that is, co-occurrence of both vector and pathogen, most of the pathogens occurred with at least one of the associated vector species. In the case of L. donovani, only a not yet confirmed vector species, P. rodhaini, could explain the occurrence of the pathogen in regions of Africa. Phlebotomus alexandri and P. longiductus on the other hand, proven vector species of L. donovani, do not seem to qualify as vectors for the pathogen. Their distribution is restricted to northern latitudes and does not match the pathogen's distribution, which lies in southern latitudes. Other more locally confined mismatches were discussed for each pathogen species. The comparative geographical GIS-overlay of vector species and pathogens functions as a first indication that testing and re-evaluation of some pathogen–vector relationships might be worthwhile to improve risk assessments of leishmaniasis.Leishmaniasis is a vector-borne disease with a broad global occurrence and an increasing number of recorded cases; however, it is still one of the world's most neglected diseases. We here provide climatic suitability maps generated by means of an ecological niche modelling approach for 32  The transmission can either be zoonotic and/or anthroponotic through the bite of an infected female phlebotomine sandfly [Phlebotomus [Leishmania species: L. major, L. tropica and L. aethiopica (being main causative parasites) as well as L. infantum and L. donovani. Visceral leishmaniasis (VL), another common and more severe form of leishmaniasis, is only associated with the Leishmania species L. infantum and L. donovani [. The specific Leishmania species cause different clinical symptoms in humans [Leishmaniasis is a vector-borne disease with a broad global distribution and an increasing number of recorded cases worldwide ,2. Howev sandfly . In Euraebotomus . Cutaneodonovani –11. The n humans ,13.A successful transmission requires the presence of pathogen, vector and host species . Thus, wLeishmania life cycle is the reservoir host [Leishmania spp., among them rock hyraxes , rodents, foxes, dogs, cats and other domestic animals [Leishmania species considered here, L. donovani and L. tropica are assumed to be anthroponotic with humans as reservoir hosts, having human-to-human transmission through the vector, but there is some evidence for the possible involvement of zoonotic transmission as well [Another important component required for the completion of the oir host . General animals . Among t as well . Althoug as well ,16.Leishmania parasite species and their associated vector species (Phlebotomus spp.) as well as their interrelationship. Most of these vector species are considered to be pathogen-specific , accounting for the distributional patterns of the five  species ), which  species , which ePhlebotomus vector species and the distributions of pathogens. For the distribution of the vector species, we used an ecological niche modelling approach to establish climatic suitability maps, whereas for the distribution of pathogens we referred to pre-existing polygon data.We focused on the comparison between the geographical distributions of 2.2.1.Phlebotomus vectors and Leishmania pathogens in Eurasia and Africa were gathered from the literature. Each vector species was linked to the pathogens it is able to transmit  approach. The occurrence data for ecological niche modelling of the Phlebotomus species was mainly obtained from [Phlebotomus species used for ecological niche modelling.Occurrence point data, and no polygon data, was available for the different ned from  and suppned from –36). Ele2.3.axent . This resolution is in accordance with the resolution of the occurrence records. Data represent averages for 1960–1990. Four temperature variables and four precipitation variables, reflecting the mean, minima, maxima and variability of annual patterns, were considered. More specifically, annual mean temperature (bio01), temperature seasonality, i.e. the standard deviation of monthly temperature (bio04), maximal temperature of the warmest month (bio05), minimal temperature of the coldest month (bio06), annual precipitation (bio12), precipitation seasonality (bio15), precipitation of the wettest month (bio13) and precipitation of the driest month (bio14) were chosen as predictive variables. These variables were considered to be ecologically relevant for the sandfly species [axent deals well with intercorrelation, so there is less need to remove correlated variables [We used the maximum entropy modelling approach implemented in the software Maxent , downlo species –41. Someariables . Other faxent default settings were made: use of only linear, quadratic and product features (cf. [axent model.The following modifications of the Mres (cf. ), maximuLeishmania pathogens and the vectors, binary climatic suitability maps were created, using equal training sensitivity and specificity threshold [To allow for a comparison of the polygonal distribution areas of the hreshold , whose ghreshold . This thhreshold .2.4.Leishmania pathogen were displayed as hatched areas  were displayed in red and the areas with the associated ed areas . Maps fo2.5.et al. [The distributional patterns of vector species indicate different climatic requirements, which are usually presented in one-variable response curves. These curves reflect the modelled niche functions (i.e. species–environment relationship) considering only one specific variable and one species. The occurrence points for the pathogens were derived from overlapping the disease occurrences provided by Pigott et al.  with theet al. ,22–24.L. donovani and its associated vector species, considering annual mean temperature.The one-variable response curves were exemplarily displayed for 2.6.axent [We used the maximum entropy modelling approach implemented in the software Maxent  , and the five pathogen species (Leishmania spp.) are summarized in Phlebotomus species, the present core distribution lies outside the considered study area, e.g. P. chinensis and P. wui with a core distribution in China. For other species, only poor distributional information is currently available . As a result, 16 out of 48 Phlebotomus species were not taken into account for further analyses due to the limited number of available occurrence records within the study area.We accounted for 48 mpetence –21,49,50Leishmania species [P. alexandri has been suggested to transmit L. donovani and L. infantum in parts of China and other countries; P. sergenti is considered a transmitter of L. tropica and L. aethiopica in parts of Ethiopia; P. duboscqi is considered vector-competent for L. major and might additionally be a competent vector for L. infantum in Gambia; P. longiductus is suspected to be vector-competent for L. infantum and for L. donovani in China. P. aculeatus is suspected to transmit L. aethiopica in Kenya according to [L. tropica according to [Most of the considered vector species are considered pathogen-specific, i.e. they are vector-competent for only one  species . However species  and the  species , a few erding to  or L. trrding to .3.2.Phlebotomus species reflect the observed occurrence distributions well . For most species, the modelled area reaches beyond the area with observed occurrences .The models of climatic suitability for all considered 32 Phlebotomus species, followed by annual mean temperature (bio01). The temperature variables generally showed higher contributions to the Maxent models than the precipitation variables, with some exceptions. In the case of P. chadii, P. langeroni, P. mongolensis, P. similis and P. transcaucasicus precipitation of the driest month (bio14) was modelled to be the first or second most important predictor variable. The permutation importance values are summarized in electronic supplementary material, table S2.Considering the average rank over all considered vector species, temperature seasonality (bio04) and minimal temperature of the coldest month (bio06) were identified as the variables best explaining the distributional patterns of 3.3.L. major, L. infantum and L. donovani overlaps greatly with areas projected to be climatically suitable for at least one of the associated vector species  and Leishmania donovani (c)). However, there are quite a few regions that have been modelled as climatically suitable for vector species without occurrence of the pathogen. Leishmania aethiopica is only known from Ethiopia and adjacent areas in Eritrea, Sudan and Kenya. Only parts of these regions are modelled to provide climatically suitable conditions for at least one of the associated vector species (d). Similarly, L. tropica matches the distributions of the associated vectors only in some areas (Eurasia and Morocco); in parts of Ethiopia, Kenya and Uganda, and in Saudi Arabia, India and Pakistan the pathogen is assumed to occur but none of the associated vector species seem to find climatically suited habitats there (e). Species-specific vector distribution patterns and observed occurrence points provide further information .The distribution patterns of the five pathogen species are largely in accordance with those of the associated vector species . The obsL. major a and Leidonovani c). Howev species d. Similats there e. Specie3.4.L. donovani and its associated vector species. The associated vector species of L. donovani might be classified into two groups P. martini , and P. rodhaini for which vector-competence has only been assumed [P. rodhaini goes beyond the area of the other species of the southern group , whereas curves for species found in more tropical climates with monotonously increasing temperature niche curves are more similar to the optimum curve of the pathogen L. donovani  values of above 0.88 for all species, the modelling results could be used as estimators for the distributional patterns of the considered vector species. For some Phlebotomus species, the area with modelled climatic suitability goes beyond the area with observed occurrence. Reasons for this could be the following: (i) a sampling bias for some of the species in the occurrence data, i.e. the species have been recorded only in some parts of its distributional range; (ii) insufficient time or dispersal barriers like oceans or mountain ranges might have prevented species to reach all places with suitable climatic conditions (so far); (iii) disregarding one or more factors others than climate being important drivers for the distributional patterns may yield an overestimation of the potential distribution.As patterns of modelled climatic suitability reflected the observed occurrences of the 32 4.2.Leishmania pathogens are in good accordance with the areas modelled to be climatically suitable for at least one of the associated vector species. However, small areas at the edges of a pathogen distribution seemingly lack suitable habitats for the associated vectors. This is most obvious for the southern edge of L. major, L. aethiopica and L. tropica. In the case of L. major, the vector distribution (proven and suspected vectors) does not entirely cover the distribution of the pathogen. The southern edge of the L. major distribution lies in the vegetation zone covered by tropical rainforest, and may be the habitat of a currently unknown vector species for this pathogen. Another reason might be an underestimation of the climatic suitability for a vector species, which was lost due to dichotomy usage. Furthermore, P. ansarii, suspected to be vector competent for L. major, but not considered here due to the lack of occurrence records, has only been reported for Iran, so far [L. aethiopica, the currently known distribution is relatively small in comparison to the other Leishmania species. Records of L. aethiopica are currently restricted to the highlands of Ethiopia and Kenya [P. aculeatus which is assumed to be vector-competent for L. aethiopica and known to occur in Kenya [P. aculeatus has been questioned by Krayter et al. [P. aculeatus and P. pedifer. Phlebotomus pedifer, as well as P. longipes, are the two main vectors for L. aethiopica [P. pedifer, 14 for P. longipes) Thus, the distribution of these species might be underrepresented in these data, which may have led to an underestimation of the area with modelled climatically suitability for these species.In general, the distributional patterns of the , so far . In the nd Kenya . The afoin Kenya . Howeverr et al. , referrithiopica , but theL. tropica is largely covered by area modelled to be suitable for P. sergenti (except the area around Kenya). Besides P. sergenti, the two vector species with suspected vector competence, P. chabaudii and P. simici, were considered in the GIS-overlay in figure 2e. The discrepancies in parts of Ethiopia, Kenya and Uganda, and in Saudi Arabia, India and Pakistan are likely to be explained by the fact that a number of known vector species  were not considered in the modelling due to lack of occurrence data. Especially for a sandfly as a species of small body size, a potential sampling bias due to an underestimation of occurrences in certain areas is quite likely.The distribution of L. major, the observed pathogen distribution area covers parts of the Sahara desert with extreme climatic conditions, hostile for many species and requiring species to be adapted. Among the seven associated vector species of L. major, P. bergeroti was the only one with modelled climatic suitability in the central parts of the Sahara.For all species, the area modelled to be climatically suitable for the vector species reaches beyond the observed pathogen distribution area. The most plausible explanation might not generally be an erroneous vector–pathogen association, but rather that the vector species is able to occur under a broader temperature range compared to the pathogen, which may require higher temperatures for its development and transmission . AnotherL. donovani and its associated vector species, which can be distinguished into two groups according to the vectors' distribution pattern: one group occurring further north in the study area  and a second group occurring further south in the study area . This separation of the associated vector species into two groups can also be found regarding the one-variable response curve with the annual mean temperature as explanatory variable. Species of the first group show optima under lower temperatures, whereas the niches of the second group, including L. donovani, show optima at warmer temperatures. The vector species of the ‘southern’ group are modelled to co-occur with L. donovani, whereas P. alexandri and P. longiductus of the ‘northern’ group do not show any overlap with the observed pathogen distribution.We had a closer look at the distributional patterns of P. argentipes, P. martini, P. orientalis and P. rodhaini is in good accordance with the observed distribution of the pathogen, i.e. the area modelled to be climatically suitable for at least one of the associated vector species covers and extends beyond the known pathogen distribution. However, distinguishing between proven and suspected vector species it becomes obvious that some parts of the observed pathogen distribution cannot be explained when only Phlebotomus species with proven vector competence  are taken into account. Phlebotomus celiae, another proven vector species for L. donovani, which has not been considered in the modelling due to the lack of occurrence data, might fill in this gap. However, P. celiae occurs in savannah and forest areas in Kenya and Ethiopia [Phlebotomus vansomerenae is also suspected to act as a vector for L. donovani in Kenya [Phlebotomus rodhaini, on the other hand, is another suspected vector and was modelled to find suitable climatic conditions in large parts of the pathogen's distribution. Based on two female individuals of P. rodhaini infected with L. donovani and caught in eastern Sudan, Elnaiem et al. [P. rodhaini being a possible vector of L. donovani for animal reservoir hosts but questioned its role in human infections. Among the considered 32 sandfly species, P. rodhaini is the only occurring vector species in some areas, and its role as an important vector seems evident. However, there may also be another disregarded vector-competent species that occurs in the region.The area modelled to be climatically suitable for the species of the second southern group with Ethiopia  and is oEthiopia . Phlebotin Kenya . Phlebotm et al.  suggeste4.3.Phlebotomus vector species and the distributions of their associated Leishmania pathogens and should lead to a better understanding of the vector–pathogen relationship, which could yield a better development of vector and disease surveillance and control programmes. However, the study is faced with some challenges, e.g. dealing with two different levels of distributional data: observed occurrence records (point data) for vector distribution and expert-based distribution areas (polygon data) for pathogen distribution. In order to bring this data into line, we have carried out an ecological niche modelling approach. In ENM approaches, some aspects should be kept in mind when interpreting the modelling results, but do not reduce the overall validity of the study. Models are only as good as the data they are based on. When using occurrence records as distributional data, one is usually confronted with a sampling bias that is also reflected in the modelling results. In some regions, the modelled vector distribution may be overestimated, as the species may be absent due to dispersal limitations (due to barriers like oceans or mountains) or due to relevant ecological factors disregarded in our approach . The inclusion of vegetation indices (e.g. enhanced vegetation index (EVI)) and soil factors as predictor variables could have improved the modelling. The disregard of species' mobility in our approach could have been partly overcome by restricting the considered areas to those accessible for the respective sandfly species. But we decided in favour of a uniform approach for all species.The purpose of the study was to compare the geographical distributions of P. langeroni, P. pedifer and P. salehli), that it could reasonably be considered insufficient for the application of ENM. We decided to keep these species in our study in order for it to be as comprehensive as possible for the vector species.Besides these limitations, the model could result in an underestimation of the vector species' distribution in other regions for several reasons. These could be due to model overfitting or due to an underrepresentation of the species’ distribution in the occurrence data (indicated e.g. by a low number of occurrence records or occurrence records being clustered in a certain region). For some species, the number of available occurrence records is so low . A poteet al. . In addiof India , but thiApart from the challenge that distributional data of different levels (point data versus polygon data) were compared, the available data refer to different time scales. Thus, the approach is based on the assumption that the spatial distribution patterns of the different components (vectors and pathogens) have not changed over time. This is a strong assumption, especially for species with low dispersal ability such as sandflies. Data availability remains a crucial limiting factor in correlative modelling approaches.5.Distribution areas of pathogens that do not overlap with areas modelled to be suitable for vector species might hint towards incomplete entomological fieldwork of vector reporting, e.g. in remotes areas. Thus, further monitoring the current distribution of sandfly species is of great importance and the joint actions of public health and veterinarian authorities in the sense of a ‘One health approach’ should be strengthened. Ecological niche modelling accounting for interactions among hosts, vectors and parasites offer powerful approaches for identifying regions potentially at risk for infection. Our approach can support risk assessments of leishmaniasis and thereby potentially help contain the disease. However, much more detail and consideration of other risk factors (e.g. human socioeconomic variables), are required for full development of risk maps . If data


Arnica chamissonis Less.) is a valuable plant species used in the pharmaceutical industry due to the content of many pharmacologically active substances and the similarity of its chemical composition to that of Arnica montana—a medicinal plant commonly used in pharmacy and cosmetics. The similarity of the two plant species implies that chamisso arnica can be a pharmaceutical substitute for the mountain arnica, i.e., an endangered and endemic plant species in Europe. Chamisso arnica extracts exhibit anti-inflammatory and antiradical activity and possesses high antioxidant properties that might be helpful in preventing or delaying the progress of free radical dependent diseases. The attributes of A. chamissonis are mainly related to the content and chemical composition of essential oil. Therefore, the objective of this study was to characterize the chemical composition of essential oil derived from A. chamissonis flower heads under a nitrogen impact. The experiment was performed on experimental fields in mid-eastern Poland on two soil types (sandy and loamy soils). The nitrogen fertilizer was applied as ammonium sulfate . Collection of flower heads was carried out in the full flowering phase, which was characterized by the highest content of essential oil. The chemical composition of essential oil was examined using GC-MS. Among the 75 ingredients of the volatile oil of chamisso arnica flower heads, alpha-pinene, cumene, p-cymene, germacrene D, spathulenol, decanal, caryophyllene oxide, beta-pinene, and benzene acetaldehyde were present at relatively high levels. Both the nitrogen application and the soil type had an effect on the oil concentration and the yield of the main constituents  with pharmacological value. Different levels of nitrogen application could be considered as a relevant way to modify the chemical composition and to increase the essential oil production.Chamisso arnica ( Melissa officinalis L. plants. Other authors reported that the nitrogen application contributed to an increase in the leaf fresh weight and the EO content in Mentha spicata L. [Mentha piperita L. [Satureja hortensis L. and Chamomilla recutita L. Rauschert [Origanum vulgare ssp. hirtum (Link) Ietswaart. Similarly, Bufalo et al. [Ocimum basilicum L., and Nurzyńska-Wierdak [Origanum syriacum L. var. aegyptiacum Tackh and demonstrated a decrease in the biosynthesis of a-terpinene and p-cymene in contrast to thymol and carvacrol. Frequently, an increase in nitrogen doses causes an increase in the EO yield due to the higher flower yield in the absence of an influence of this nutrient on the EO concentration in tissues, as demonstrated in the case of Matricaria recutita L. [Essential oils (EO) are important secondary metabolites of plants. They have been used for centuries not only in different fields/branches of industry but also in ethnobotanical medicine . The quaicata L. , the dryerita L. , as wellauschert ,22. On tauschert  showed ao et al.  did not -Wierdak  found th-Wierdak , who dem-Wierdak  showed autita L. .Arnica chamissonis Less.) is an herbaceous perennial herb and a medicinal plant that is widely used as an herbal remedy. Its occurrence range extends from the Alaskan Archipelago south to the San Bernardino Mountains in California and from the southern Rocky Mountains to New Mexico [A. chamissonis are mainly related to the content and chemical composition of EO. Given its content of numerous pharmacologically active substances [Arnica montana, i.e., a medicinal plant rich in secondary metabolites [A. chamissonis seeds than extracts of A. montana seeds. Either alone or in combination with other active compounds, the former can be used in cosmetic, nutraceutical, and pharmaceutical applications. It should also be stressed that A. montana does not show yield stability in the conventional European agricultural conditions, as shown by field trials conducted in France, Germany, and Switzerland, in contrast to A. chamissonis [A. chamissonis a suitable species to substitute A. montana for pharmaceutical purposes [A. chamissonis raw material and introduction of this plant species to cultivation tests on different soil types can help to reduce the pressure on the endangered and highly valued medicinal A. montana [Chamisso arnica  to evaluate the influence of different nitrogen doses and soil type on the quantity, chemical composition, and yield of EO in the flower heads of A. chamissonis.While analyzing the aforementioned examples of the role of nitrogen in crop generation and modification of the chemical composition of EO in medicinal and aromatic plant species, it can be expected that the modification of the nitrogen level may result in a higher EO yield and new EO quality in the case of r demand , a new cfficient ,29; therfficient ,58,59,60A. chamissonis and the EO yield under nitrogen fertilization applied into the two different soil types are presented in −1 resulted in an increased crop yield of A. chamissonis flower heads from 1210 to 1941 kg ha−1 and from 994 to 1563 kg ha−1 on the loamy (L) and sandy (S) soils, respectively.The content of EO in the flower heads of −1 also resulted in enhanced accumulation of EOs in the A. chamissonis flower heads . A further increase in the N dose up to 120 kg ha−1 had no effect on the content of volatile oils in the A. chamissonis flower heads in any soil type. The N fertilization differentiated the EO yield significantly , i.e., by ca. 90% and by ca. 86%, respectively. The EO content and yield were higher in the raw material taken from plants growing on the L soil than on the S soil at each fertilization level. Moreover, the level of the essential oil yield in A. chamissonis flower heads cultivated without fertilization on the L soil was comparable with that on the S soil at the 30 kg ha−1 N rate.The increase in the N rate from 0 to 90 kg haficantly . An incrA. chamissonis flower heads , an over 2-fold higher yield of alpha-pinene, cumene, germacrene D, beta-pinene, and p-cymene and over 12-fold higher amounts of benzene were recorded in the case of flower heads of arnica plants growing on the L soil in comparison to the plants from S soil. In contrast, the spathulenol yield was higher in plants growing on the S soil than on the L soil. The yield of decanal and caryophyllene oxide in the control samples was similar in the raw material obtained from the two soil types. In the case of plants cultivated on both L and S soils, the increase in the N dose caused an increase in the yield of the EO components.Among the nine main components of the volatile oil from the ceeds 3% , alpha-pceeds 3% . There w−1 on the S soil resulted in a higher yield of alpha-pinene (from 196.82 to 542.30 g ha−1), cumene (from 149.37 to 252.92 g ha−1), germacrene D (from 74.55 to 153.68 g ha−1), spathulenol (from 101.23 to 129.13 g ha−1), decanal (from 85.27 to 112.66 g ha−1), caryophyllene oxide (from 77.57 to 126.84 g ha−1), and beta-pinene (from 30.53 to 78.23 g ha−1). An increase in the N dose to 60 kg ha−1 on this soil type resulted in an increase in the p-cymene yield from 70.83 to 183.20 g ha−1; however, a further increase in the N dose caused a statistically significant decrease in this EO component. In the case of benzene acetaldehyde, the yield increased with the increase in the N dose to 90 kg ha−1, whereas the increase in the N dose to 120 kg ha−1 did not cause a significant increase in the yield.The increase in the N rate from 0 to 120 kg ha−1 resulted in a growing yield of cumene (from 205.52 to 366.10 g ha−1), p-cymene , beta-pinene (from 65.96 to 122.15 g ha−1), and benzene acetaldehyde (from 62.49 to 147.21 g ha−1). In the case of alpha-pinene, the N dose 90 kg ha−1 caused an increase in the yield from 482.65 to 894.17 g ha−1, whereas the application of 120 kg N ha−1 did not cause a further increase in the yield. The yield of the other main components of the volatile oil of A. chamissonis growing on the L soil also increased under the N fertilization: germacrene D (from 90.74 to 161.82 g ha−1), spathulenol (from 73.79 to 139.90 g ha−1), decanal (from 81.63 to 130.85 g ha−1), and caryophyllene oxide (from 72.52 to 154.86 g ha−1). Of note, the N dose of 120 kg ha−1 caused a statistically significant decrease in these EO components  G. Don [A. montana populations have shown chemical diversity [Seventy-five compounds constituted from 93.91 to 99.96% of the total EO in the flower heads in thium L.  or Achilolium L. . Howeversum spp.  and Tanalgare L. . The comć et al. , Kowalskć et al. . In our ć et al. , boron fć et al. . It shouć et al. . On the i et al. , who invlefolium , A. absisinthium , Artemisgaris L. , or Heli) G. Don . Similariversity ,45,67.Centaurea spp. [Eupatorium intermedium DC. [Copaifera langsdorffii Desf. [Cupressus sempervirens L. cone EO and Pistacia vera L. [Santolina africana Jord. et Fourr. [Senecio vernalis Waldst. & Kit. grown in Turkey [As demonstrated in this paper, the nitrogen application modified the EO composition. However, very interesting is the fact that the proportion  of the main EO components from the flower heads of this species, i.e., alpha-pinene, caryophyllene oxide, and germacrene D, is similar to the chemical oil profile in such species as rea spp. , Eupatordium DC. , and Copii Desf. . Alpha-p vera L. ,82. In tt Fourr. . Alpha-pn Turkey .A. chamissonis genotype was cultivated in the same site and on the same sandy soil, and the flower heads were collected in the full flowering phase, which is characterized by the highest content of EO, but in different weather conditions. Probably, this factor, regarded as a determinant of the quantitative and qualitative EO composition [In the present study, monoterpene hydrocarbons were the most abundant group, representing from 47.01 to 53.74% in the EO obtained from plants cultivated on the L soil and from 35.71 to 48.21% in EO obtained from plants cultivated on the S soil. The second largest group comprised sesquiterpene hydrocarbons, which accounted for 10.96–13.87% and 11.65–19.15% in the EO obtained from plants cultivated on the L and S soil, respectively. Additionally, based on the comparison of the control samples from the two soil sites, it can be concluded that L soils, which are rich in macroelements and are more fertile than S soils, favor production of monoterpene hydrocarbons rather than sesquiterpene hydrocarbons. In this case, the habitat characteristics ,17 seem position , had theA. chamissonis flower heads with content exceeding 3%, there was a relatively high yield of alpha-pinene, cumene, p-cymene, germacrene D, spathulenol, decanal, caryophyllene oxide, beta-pinene, and benzene acetaldehyde. It is worth emphasizing that, already in the control sample (0 kg N ha−1), the yield of alpha-pinene, cumene, germacrene D, beta-pinene, and p-cymene was over 2-fold higher. The yield of benzene acetaldehyde was over 12-fold higher in the EO of flower heads in plants growing on the L soil in relation to the S soil; in contrast, the spathulenol yield was higher in plants growing on the S soil than the L soil. The yield of decanal and caryophyllene oxide in the control samples was similar in the raw material obtained from the two soil types. Both in the cases of plants cultivated on the L soil and on the S soil, the increase in the nitrogen dose caused an increase in the yield of the EO components.Among the nine main ingredients of the volatile oil of −1 in the S soil resulted in a higher yield of some components of EO, including alpha-pinene, cumene, germacrene D, spathulenol, decanal, caryophyllene oxide, and beta-pinene. In turn, the increase in the N dose to 60 kg ha−1 and 90 kg ha−1 in this type soil caused an increase in the p-cymene and benzene acetaldehyde yields, respectively.The present study showed that the increase in the N rate from 0 to 120 kg haA. chamissonis flower heads, as in the case of boron fertilization [The successive N fertilization caused an increase in the alpha-pinene concentration in the EO of lization . TherefoA. chamissonis EO determine the biological activity of formulations derived from this plant raw material. There are sparse data on A. chamissonis volatile oils despite the fact that the EO contributes to all medicinal properties of the plant. Alpha-pinene, germacrene D, p-cymene, and caryophyllene oxide are the main components of essential oils in Arnica spp. [A. chamissonis EO is the primary compound present in numerous medicinal plant species belonging to the Asteraceae family, e.g., Chrysanthemum coronarium L. [Eupathorium buniifolium Hook. Ex Hook & Arn. [Monticalia greenmaniana (Hieron) C. Jeffrey [Ducrosia anethifolia Boiss [A. chamissonis raw material and the high yield of this compound, especially under the nitrogen fertilization, make this plant very interesting as a source of important secondary metabolites that can be produced in field conditions, but definitely on very fertile nutrient-rich soils with a granulometric composition of clay. Decanal is another compound exhibiting antibacterial [A. chamissonis oil. It is one of the main components found in some plant species, e.g., A. montana [The compounds present in the ica spp. ,17. Alphica spp. , anti-inica spp. , antimicica spp. ,85, antiica spp. , anticanica spp. , and antica spp.  propertiarium L. , Eupathok & Arn. , and Mon Jeffrey , or repria Boiss . Moreoveia Boiss . Alpha-pia Boiss . The higacterial , antioxiacterial  activiti montana ,17. Cary montana ,47. This montana ,49,50.A. millefolium [A. vulgaris [E. intermedium [M. greenmaniana [S. africana [Cananga odorata Hook. F. & Thomson flowers [C. odorata flowers is an important raw material for the cosmetic and perfume industry [A. chamissonis flower heads, especially under N fertilization, makes the plant species attractive for use in the cosmetic and perfume industry. Given the similarity of the chemical composition of oils characteristic for both arnica species , it can be suggested that the analyzed group of secondary metabolites should exhibit common biological properties, and both boron [A. chamissonis flower heads and essential oil-generating factors.Germacrene D is a nonoxygenated sesquiterpene present as a dominant component in the EO of plant species in the Asteraceae family, e.g., lefolium , A. vulgvulgaris , E. inteermedium , M. greenmaniana , and S. africana . It is wafricana , were alafricana , antifunafricana , antibacafricana , antioxiafricana . This bi flowers ,90 with th boron  and nitrArnica chamissonis Less. species used in the experiment was identified by Anna Rysiak, a taxonomist from the Maria Curie-Skłodowska University in Lublin based on the reference material from the collection of medicinal plants of the Department of Industrial and Medicinal Plants of the University of Life Sciences in Lublin. In 2017, the raw material for the chemical analyses was taken from 2-year-old A. chamissonis individuals from two experimental fields at the University of Life Sciences in Lublin located in the eastern part of Poland. The first experimental field  was located on sandy (S) soil , and the other experimental field  was located on loamy (L) soil . The sandy soil was characterized by moderate content of organic matter , moderate phosphorus , low potassium , and very low magnesium levels , with very acidic reaction. In turn, the loamy soil was characterized by high content of organic matter (1.88%), very high phosphorus (258 mg kg−1), high potassium (39 mg kg−1), and moderate magnesium levels (51 mg kg−1), with neutral reaction.The A. chamissonis flower heads were collected in the full flowering phase. This phase is characterized by the highest content of EO in the flower heads [er heads . The floTwenty grams of powdered chamisso arnica inflorescences were submitted to water-distillation in a Deryng apparatus with 500 mL water for 3 h according to the Polish Pharmacopoeia VI . The metThe chromatographic analysis was performed according to procedures described previously ,17. The 12 and C19) according to procedures described by Kowalski and Wawrzykowski [The qualitative analysis was carried out on the basis of MS spectra, which were compared with the spectra of the NIST library  and withzykowski .p < 0.05. The statistical analyses were carried out using the Statistica 6.0 software. Principal component analysis (PCA) was applied in order to explain the relationships between oil ingredients and to show variability factors. Prior to the PCA, the data on the EO content and oil yield of the main components (over 3%) were centered. The analyses were carried out using the statistical package (MVSP) program version 3.1. [The two-way analysis of variance (ANOVA) and subsequent Tukey’s tests were used. The differences were considered significant at ion 3.1. .Arnica chamissonis are mainly related to the content and chemical composition of essential oil. Among the 75 ingredients of the essential oil from A. chamissonis flower heads, alpha-pinene, cumene, p-cymene, germacrene D, spathulenol, decanal, caryophyllene oxide, beta-pinene, and benzene acetaldehyde were characterized by relatively high levels. Both the nitrogen application and the soil type, which had not been tested so far, had an effect on the concentration and yield of the main constituents, such as alpha-pinene, decanal, and germacrene D, i.e., molecules of high pharmacological value. Different levels of nitrogen application can be considered as a relevant way to modify the chemical composition and to increase the raw material and essential oil production. In further studies of factors modifying the EO chemical composition and influencing the EO concentration and yield of the main and very important EO compounds, attention should be paid to the simultaneous use of nitrogen and other microelements to achieve synergistic results. The flower head yield, EO concentration, and essential oil yield obtained in the presented study show that the climatic conditions of mid-eastern Poland are favorable for A. chamissonis and a wide range of pharmacologically active substances can be produced by this plant species in this region of Europe.The attributes of


Mycobacterium ulcerans is the causative agent of Buruli ulcer, also known in Australia as Daintree ulcer or Bairnsdale ulcer. This destructive skin disease is characterized by extensive and painless necrosis of the skin and soft tissue with the formation of large ulcers, commonly on the leg or arm. To date, 33 countries with tropical, subtropical and temperate climates in Africa, the Americas, Asia and the Western Pacific have reported cases of Buruli ulcer. The disease is rarely fatal, although it may lead to permanent disability and/or disfigurement if not treated appropriately or in time. It is the third most common mycobacterial infection in the world after tuberculosis and leprosy. The precise mode of transmission of M. ulcerans is yet to be elucidated. Nevertheless, it is possible that the mode of transmission varies with different geographical areas and epidemiological settings. The knowledge about the possible routes of transmission and potential animal reservoirs of M. ulcerans is poorly understood and still remains patchy. Infectious diseases arise from the interaction of agent, host and environment. The majority of emerging or remerging infectious disease in human populations is spread by animals: either wildlife, livestock or pets. Animals may act as hosts or reservoirs and subsequently spread the organism to the environment or directly to the human population. The reservoirs may or may not be the direct source of infection for the hosts; however, they play a major role in maintenance of the organism in the environment, and in the mode of transmission. This remains valid for M. ulcerans. Possums have been suggested as one of the reservoir of M. ulcerans in south-eastern Australia, where possums ingest M. ulcerans from the environment, amplify them and shed the organism through their faeces. We conducted a systematic review with selected key words on PubMed and INFORMIT databases to aggregate available published data on animal reservoirs of M. ulcerans around the world. After certain inclusion and exclusion criteria were implemented, a total of 17 studies was included in the review. A variety of animals around the world e.g., rodents, shrews, possums (ringtail and brushtail), horses, dogs, alpacas, koalas and Indian flap-shelled turtles have been recorded as being infected with M. ulcerans. The majority of studies included in this review identified animal reservoirs as predisposing to the emergence and reemergence of M. ulcerans infection. Taken together, from the selected studies in this systematic review, it is clear that exotic wildlife and native mammals play a significant role as reservoirs for M. ulcerans. Mycobacterium ulcerans in 1896. Later, in the late 1930s, two general practitioners, Drs. J. R. Searl and D. G. Alsop, working in rural Victoria, Australia, noticed a group of cases of mysterious skin ulcers around the town of Bairnsdale [M. ulcerans as the causative organism of the mysterious skin ulcer [M. ulcerans infection was identified in the Buruli County of Uganda  in the 1960s and the disease was termed ‘Buruli ulcer’ (BU) thereafter [Sir Albert Cook, a British missionary doctor appointed at the Mengo Hospital in Kampala, Uganda, first noted the skin ulcer caused by in ulcer . The firM. ulcerans are prevalent in Australia only, where several cases of BU have been described in both native wildlife and domestic mammal species such as koalas (Phascolarctos cinereus) [Pseudocheirus peregrinus) [Trichosurus cunninghami) [M. ulcerans in Victoria [There have been several known outbreaks of Buruli ulcer around the world and each outbreak has its own unique characteristics in terms of epidemiology and the animals reported to be involved in transmission ,5. The Winereus) ,13, commegrinus) ,15, a moinghami) ,15, two inghami) , an alpainghami) , four doinghami)  and a cainghami) . Recent Victoria .Mycobacterium ulcerans’) AND , accessed on 6 May 2018. The title and abstract of each of the articles were initially scanned to ensure that the included articles met the aim and scope of the systematic review. Articles that were deemed irrelevant to the aim of this systematic review or out of the research scope were excluded. For those articles that were not clear by the title and abstract, the full text was retrieved and further analyzed in order to determine if they met the inclusion and exclusion criteria below. The studies that reported only experimental or laboratory exposure of M. ulcerans in animals were excluded. The search strategy exclusively focused on potential animal reservoirs, not the vectors. The detection of the causative agent had to be confirmed by culture of bacteria and/or PCR. To be considered positive a sample needed to be confirmed either by culture of bacteria or positive for IS 2404 and reconfirmed by KR and IS 2606. Undoubtedly, PCR targeting IS 2404 is highly specific for detecting M. ulcerans in clinical specimen [M. ulcerans from environmental samples, confirmatory PCR targeting two additional insertion sequences, IS 2606 and the ketoreductase B domain (KR), is essential to differentiate M. ulcerans from other environmental mycobacteria that may carry IS 2404 and other non-mycolactone-producing mycobacteria [M. ulcerans. There were no language restrictions. Risk of bias was assessed by one reviewer on the basis of independent factors such as sample size, location and nature of infection. The PRISMA guidelines developed by the Centre for Review Dissemination (CRD) were used as the methodology for the systematic review . A reviespecimen . Howeverbacteria . Thus, IThe total number of discovered articles in MEDLINE database was 351. Three hundred and fourteen articles were excluded after reading the title and abstracts as they were not relevant to the research question. Full texts of thirty-seven studies were retrieved in portable document format (PDF) for further analysis. Of these remaining 37 studies, 19 were excluded as they clearly did not meet inclusion criteria . One additional duplicate article was excluded as well. The remaining 17 studies from the PubMed database were included for systematic review. There were no additional articles in INFORMIT that did not appear in the initial MEDLINE search results. The flow chart for study selection process is shown in Out of the 17 included studies, ten were conducted in Australia, two in Ghana and one was conducted in each of Ivory Coast, North America, United States, Benin and Japan. The basic characteristics of selected studies for review are shown in M. ulcerans around the world recorded to date. This is essential for understanding the epidemiology and mode of transmission of the disease, which subsequently aids in prevention, control and elimination strategies.This systematic review assessed the potential animal reservoir of M. ulcerans infection in four koalas in 1980 at Raymond Island, Australia [M. ulcerans. Diagnosis was made on pathological and bacteriological examination; the PCR-based method used for the identification of M. ulcerans from clinical and environmental samples was only implemented in 1996 [M. ulcerans in Australia have been reported in marsupial species such as koalas [M. ulcerans DNA [M. ulcerans DNA in this study, suggesting terrestrial mammals such as possums are potential reservoirs of M. ulcerans in southeast Australia. Several studies have identified possums (both ringtail and brushtail) as potential reservoirs since then [M. ulcerans in Victoria, a study conducted by Roltgen and colleagues (2013) in northern Queensland, Australia, detected M. ulcerans DNA from two bandicoot faecal samples, suggesting the possibility that bandicoots are a potential reservoir of M. ulcerans in Far North Queensland [Out of 17 studies included in this review, 10 were conducted in Australia. In Australia, the disease is more prevalent in the southeastern state of Victoria and in Far North Queensland. After the detection of ustralia , the ent in 1996 . Non-hums koalas , ringtais koalas ,15,31, hs koalas , alpacass koalas  and catsrans DNA . Only 1%M. ulcerans, but no specific DNA was detected from any of their samples [Thryonomys swinderianus (agouti) were reported positive for M. ulcerans in a study conducted by Bi Diangoné Tian and colleagues (2014) from the Ivory Coast [M. ulcerans in Africa. However, RT-PCR targeting IS 2606 was not conducted to confirm M. ulcerans. A faecal survey of domestic animals in rural Ghana for M. ulcerans conducted by Tobias and associates suggested no evidence of association between domestic animals and M. ulcerans in endemic and non-endemic villages in Ghana [M. ulcerans-positive DNA or cases in non-human species, suggesting that transmission dynamics may be different in Africa and Australia or, alternatively, a host animal is yet to be identified in Africa.Out of the 17 studies included in this review, four were conducted in West African countries: two in Ghana ,25, one  samples . Despitery Coast . They suin Ghana . Unlike M. ulcerans DNA or cases in non-human species in any country other than Australia. A study conducted by Heckert in 1997 at Chesapeake Bay, USA detected a new Mycobacterium species from wild striped bass [M. marinum, M. ulcerans, and M. tuberculosis. Similarly, Sakaguchi and associates reported an atypical mycobacterial infection in an Indian flap-shelled turtle (Lissemys punctata punctata), imported from India to Japan in an aquarium [rpoβ gene revealed the isolate had 89–100% homology to M. ulcerans and M. marinum. Again, this study could not differentiate M. ulcerans from mycolactone-producing M. marinum (MPMM). Appleyard and Clark (2002) reported a new Mycobacterial species, namely ‘Mycobacterium visibilis’ from three cats initially suspected of having M. ulcerans infection [No study has reported ped bass . This neaquarium . A PCR anfection .M. ulcerans from clinical and environmental samples, there has been a significant increase in overall knowledge of BU. There is no record of direct human-to-human transmission of M. ulcerans, unlike tuberculosis and leprosy. Australia is the only country where non-human cases of BU have been identified, with small mammals, especially possums and, to some extent, bandicoots, being implicated as potential reservoirs of M. ulcerans. Despite there having been several outbreaks in African countries, no non-human cases have been recorded so far and there is no evidence of any animal acting as a potential reservoir for this organism. None of the studies included in this review discussed strain variation of M. ulcerans in different geographical regions leading to an increase or decrease in susceptibility among animal or human population. Compared to other mycobacteria, such as M. tuberculosis, there is very little genetic diversity among isolates of M. ulcerans. Some variation among the strains of M. ulcerans from Africa, the Americas, Asia and the Western Pacific has been recorded; however, the linkage between these various strains and virulence in human or animal population has not been recognized so far. Remarkable differences in the type of mycolactone produced by M. ulcerans in different geographical location has been recorded. African strains produce more mycolactone variant A and B, whereas strains from Australia produce more mycolactone variant C. However, this variation has nothing to do with host susceptibility to M. ulcerans; rather, it determines cytopathogenecity and thus clinical presentation of disease.Human cases of BU have been reported in more than 30 countries from Africa, America, Asia and Oceania. Since the implementation of PCR-based methods for the detection and identification of This systematic review suggests the need for extensive laboratory and field research focusing on domestic animals and wildlife to elucidate their roles in BU-endemic countries.


Pyrus hopeiensis is a valuable wild resource of Pyrus in the Rosaceae. Due to its limited distribution and population decline, it has been listed as one of the “wild plants with a tiny population” in China. To date, few studies have been conducted on P. hopeiensis. This paper offers a systematic review of P. hopeiensis, providing a basis for the conservation and restoration of P. hopeiensis resources. In this study, the chloroplast genomes of two different genotypes of P. hopeiensis, P. ussuriensis Maxin. cv. Jingbaili, P. communis L. cv. Early Red Comice, and P. betulifolia were sequenced, compared and analyzed. The two P. hopeiensis genotypes showed a typical tetrad chloroplast genome, including a pair of inverted repeats encoding the same but opposite direction sequences, a large single copy (LSC) region, and a small single copy (SSC) region. The length of the chloroplast genome of P. hopeiensis HB-1 was 159,935 bp, 46 bp longer than that of the chloroplast genome of P. hopeiensis HB-2. The lengths of the SSC and IR regions of the two Pyrus genotypes were identical, with the only difference present in the LSC region. The GC content was only 0.02% higher in P. hopeiensis HB-1. The structure and size of the chloroplast genome, the gene species, gene number, and GC content of P. hopeiensis were similar to those of the other three Pyrus species. The IR boundary of the two genotypes of P. hopeiensis showed a similar degree of expansion. To determine the evolutionary history of P. hopeiensis within the genus Pyrus and the Rosaceae, 57 common protein-coding genes from 36 Rosaceae species were analyzed. The phylogenetic tree showed a close relationship between the genera Pyrus and Malus, and the relationship between P. hopeiensis HB-1 and P. hopeiensis HB-2 was the closest. Pyrus belongs to the Pyrus ssp. of the Maloideae subfamily (Rosaceae), which mainly includes temperate fruit trees. There are more than 30 species in this genus, and 13 species are present in China P. hopeieingzhong  revealedPyrus. The pollen morphology of P. sinkiangensis is similar to that of the Western pear, indicating a close relationship [P. calleryana has many primitive characteristics, and it is a primitive species of Pyrus in China. The pollen morphology of P. bretschneideri, also present in China, has the characteristics of both P. pyrifalia and P. ussuriensis, and may be a natural hybrid of P. pyrifalia and P. ussuriensis. Cytological markers enabled the analysis of the number, banding, karyotype, and meiosis behavior of the chromosomes. P. phaeocarpa has a similar karyotype to that of P. betulifolia, and those of P. sinkiangensis, P. hopeiensis, and P. serrulata were also similar [P. bretschneideri and P. pyrifolia were closely related. However, these methods have few characteristic sites, poor polymorphism, and low accuracy, and provide a limited amount of information. To date, the relationships among Pyrus species, their origin, evolution of cultivation systems, and the origin of some suspicious species and hybrids remain unclear.Pollen morphological identification, cytological markers, isozymes, and other methods have also been studied with a view to classifying tionship . The pol similar ,33. In 1 similar  proved, Pyrus. However, there remain some deficiencies in the study of the interspecific relationships and origins of hybrids. Results based on random amplification of polymorphic DNA (RAPD) showed that the origin of P. sinkiangensis involved the crossing of many Eastern and Western pear species and that the genetic relationship between P. bretschneideri and P. pyrifolia is very close [P. hopeiensis, P. betulifolia and P. phaeocarpa were closely related to each other. In the same way, P. phaeocarpa is considered to be a hybrid of P. betulifolia and P. ussuriensis, whereas P. hopeiensis is a hybrid of P. phaeocarpa and P. ussuriensis. In a study using RAPD, P. hopeiensis and P. phaeocarpa shared some spectral bands with P. betulifolia and P. ussuriensis [P. ussuriensis and P. hopeiensis using internal transcribed spacer (ITS) sequences, which is consistent with the results in our study [In recent years, molecular markers based on DNA, such as restriction fragment length polymorphisms (RFLPs) and simple sequence repeats (SSRs) have been used to investigate the genetic relationships, genetic diversity, and germplasm of ry close . RAPD, iuriensis ,37,38. Zur study .trnS-psbcc, trnL-trnF and accD-pasI) [Pyrus. Phylogenetic trees based on combinations of the sequences of trnL-trnF and accD-psaI in the chloroplast non-coding regions have further confirmed the theory of an independent evolution of the Oriental pear and the Western pear from the background of matrilineal evolution, and have shown the close relationship between P. bretschneideri and P. pyrifolia [trnL-trnF region of cpDNA showed that P. sinkiangensis is closely related to the Western pear and the Oriental pear; the relationship between P. betulifolia and P. ussuriensis is close; P. bretschneideri is a hybrid of P. ussuriensist, P. phaeocarpa, and P. pyrifolia; and that the Western pear and Oriental pear are related to each other [Pyrus. However, no comprehensive and systematic cpDNA sequence analysis of Pyrus exists in China. To further our understanding of the inter-species relationships of Pyrus and to reveal the origin of hybrids and explore the evolutionary model of Eastern and Western pears, a wider range of representative species and varieties of Eastern and Western pears must be selected, and the nuclear gene fragments inherited by their parents should be combined, especially the low copy nuclear gene introns.Because the chloroplast genome is the second-largest genome after the nuclear genome, it is maternally inherited in most angiosperms; thus it reflects the maternal evolutionary history, and this helps us to understand the maternal ancestors of suspected hybrids. The coding and non-coding regions of the chloroplast genome evolve at different rates, making them suitable for systematic research at different levels. The coding region is highly conserved and is only suitable for phylogenetic studies of families, orders, and higher taxonomic levels, whereas the non-coding regions are less constrained by function and the rapid evolutionary rate is suitable for plant phylogenetic studies at interspecific and subspecies levels. At present, the successful design of a set of universal primers for the chloroplast gene non-coding regions (such as cD-pasI)  has madeyrifolia . A studych other . The seqP. hopeiensis HB-1, P. hopeiensis HB-2, and three local Pyrus species, P. ussuriensis Maxin. cv. Jingbaili (which belongs to the P. ussuriensis family), P. communis L. cv. Early Red Comice  and P. betulifolia (most widely used in northern China as pear rootstocks) in Changli, Hebei Province and were stored before being transported to ORI-GENE Ltd., a science and technology company based in Beijing, China, for chloroplast genome sequencing.In early May 2017, fresh leaves were collected from The total DNA of fresh young leaves was extracted using a plant DNA extraction kit . Agarose gel electrophoresis was used to detect DNA integrity, and purity and concentration were ascertained. The Illumina HiSeq platform was used to sequence the total DNA. After sequencing, the raw data was initially screened to remove low quality regions affecting the data quality and subsequent analysis needed to obtain the expected clean data. The SOAPdenovo2.01  oligonuchttp://ogdraw.mpimp-golm.mpg.de/index.shtml) based on the annotated results.CpGAVAS  was usedhttps://sourceforge.net/projects/kakscalculator2) was then used to calculate Ka/Ks, which was used to analyze the selection pressure on different Pyrus species during the evolutionary process. Chloroplast genome sequences of 36 Rosaceae species were selected from NCBI and 57 common protein coding genes were used to explore the evolution of the Pyrus chloroplast genome, using Arabidopsis thaliana as the outgroup. The taxonomic status was confirmed. The annotated files of all of the genomes were downloaded from NCBI and the protein sequences of any genes shared among the chloroplast genomes of all of the species were extracted. Each gene was placed in a file in which each genome contained only one protein sequence. MUSCLE was used to make multiple sequence alignments for each file. The first and last sequences were aligned according to the genome source to obtain a growing alignment sequence: final.fa. MEGA7.0 software was then used to construct a neighbor-joining tree and the CGView Server was used to analyze the genetic variation among the chloroplast genomes of the five Pyrus species.The protein and coding sequences (CDS) of each sample were extracted from the annotated files of each sample and the pairwise protein sequences aligned using MUSCLE software. The aligned protein sequences were converted to DNA sequences using PAL2NAL. KaKs_Calculator2.0  softwareP. hopeiensis chloroplast genomes. The length of the chloroplast genome of P. hopeiensis HB-1 is 159,935 bp, which is 46 bp longer than that of P. hopeiensis HB-2. The SSC and IR regions of the two Pyrus genotypes were the same length, with the only difference present in the LSC region. A total of 118 genes were identified in P. hopeiensis HB-1, and it only lacked the MATK protein-coding gene that was associated with biosynthesis in P. hopeiensis HB-2. The GC content of P. hopeiensis HB-1 was only 0.02% higher than that of P. hopeiensis HB-2. A total of 11 genes in the chloroplast genome of P. hopeiensis HB-1 contained introns, and an additional trnI-TAT gene not present in P. hopeiensis HB-2. ycf3 is the only gene that contained two introns. The chloroplast genome structure and size, gene species, gene number, and GC content of P. hopeiensis were similar to those of the other three Pyrus species investigated. Almost all of the protein coding sequences and amino acid codons showed an obvious codon preference. Selection pressure analysis revealed that the chloroplast genomes of different pears were affected by different environmental pressures during the evolutionary process, which may account for the differences in gene numbers among the five Pyrus species. Phylogenetic analysis strongly supports the status of Pyrus in the Rosaceae. This study adds to our knowledge of the molecular evolution of Pyrus, and will be of use for the genetic breeding and chloroplast engineering of Pyrus.In this study, we reported the de novo sequencing results of


The authors apologise for the following error:A. sylvaticus used in the study was “derived from wild-caught animals collected from a woodland in the Wirral, UK around 5 years ago”.It was mistakenly stated that the colony of et al., 2010), and then a subset of the wood mice was brought to the University of Edinburgh from Liverpool around 6 years ago.Rather, the outbred colony was originally established by J. Clarke in 1995 at the Department of Zoology, University of Oxford, and was maintained with occasional introductions of wild wood mice. The wood mouse colony moved to the University of Liverpool, Faculty of Veterinary Science around 10 years ago  Pathogenesis of a Model Gammaherpesvirus in a natural host. Journal of Virology84(8), 3949–3961.In addition, an update is available for a reference that was in preparation at the time of publication :Clerc M, Babayan SA, Fenton A and Pedersen AB (2019) Age affects antibody levels and anthelmintic treatment efficacy in a wild rodent. International Journal for Parasitology: Parasites and Wildlife8, 240–247. DOI: 10.1016/j.ijppaw.2019.03.004


Scientific Reports 10.1038/s41598-018-32694-7, published online 26 September 2018Correction to: In the original HTML version of this Article, H. M. Ogmundsdottir was incorrectly listed as one of the corresponding authors. The correct corresponding authors for this Article are M. H. Ogmundsdottir and E. Steingrimsson. Correspondence and request for materials should be addressed to mho@hi.is or eirikurs@hi.is. This error has now been corrected in the HTML version of the Article; the PDF version was correct from the time of publication.


Leishmania spp. are known to cause disease in man and animals. Rats are considered important reservoir hosts and transmission takes place through the bite of female sand fly, Phlebotomus spp. To the best of our knowledge, there is no published information on Leishmania infection in rats in Grenada. This study was conducted to estimate the antibodies for visceralizing Leishmania spp. (VL) in rats (Rattus norvegicus) from Grenada.R. norvegicus) were trapped live from two parishes (St. George and St. David) in Grenada. Following anesthesia, blood was collected from the heart through thoracic puncture. The serum was collected after the centrifugation of blood. Serum was tested for antibodies to VL. with a commercially available immunochromatographic dipstick test which is licensed for use in animals and humans.A total of 146 brown rats . No significant differences were found between sexes and young or adults. The prevalence between parishes (St. George and St. David) was also not significant.The seroprevalence of antibodies against R. norvegicus) in Grenada are exposed to Leishmania spp. The rats could play an important role in the transmission of leishmaniasis to humans and other animals in Grenada.The results show that rats ( Leishmania. Leishmania spp. are insect-vectored intracellular protozoan parasites causing disease in humans and animals  in Grenada) was approved by the Institutional Animal Care and Use Committee (IACUC # 16009-R) of the St. George’s University, Grenada.The project  with cheese and various local fruits as bait. Attempts were made to trap the rats from and near the residential buildings. Traps were placed 2 days per week in the evening and visited next day morning. Traps with rats were covered with black cloth and transported to the necropsy laboratory of the School of Veterinary Medicine and transferred to the anesthesia machine. Rats were anesthetized using isoflurane in oxygen through portable vet anesthesia machine isoflurane vaporizer VET CE., manufacturer DRE et al. [The anesthetized rats were examined for their physical health and weighed. Gender was also recorded. Rats <100 g were grouped as young and over 100 g as adult, following the methodology used by Panti-May et al. . Blood wSera were separated from the blood by centrifugation at 1500 g for 15 min at room temperature and stored at −80°C till tested.Leishmania spp. with a commercially available immunochromatographic (ICT) dipstick test which is licensed for use in animals and humans . The qualitative ICT is based on recombinant antigen K39, an immunodominant amastigote protein, to detect antibodies specific to VL species [Rats were tested for antibodies to visceral  species . The ICT species ,15. Rat http://www.graphpad.com/quickcalcs/contingency2).The data were analyzed by the Fisher’s exact test, using GraphPad statistical software (Leishmania spp. were detected in 34 of 146 brown rats (23.3%) (CI 95% from 16.70 to 30.99). The serological results according to parish, sex and age are presented in Antibodies to R. norvegicus. Di Bella et al. [R. norvegicus in Italy using indirect immunofluorescent techniques. In the same study, they found 57.55% seropositivity by IFA and 45% by molecular technique in Rattus rattus. A much lower prevalence of Leishmania spp. in 1 of 16 (6.25%) in R. norvegicus in Greece was found by Papdogiannakis et al. [et al. [R. rattus in Brazil using ELISA test. However, they found a lower prevalence with indirect fluorescent antibody test (IFAT) (3.3%) and polymerase chain reaction (PCR) (7.1%). The results of previous researchers show that variation in the prevalence of Leishmania spp. in rats (R. rattus and R. norvegicus) differs with different diagnostic techniques and in different geographic regions.There are limited studies on leishmaniasis in a et al.  reporteds et al. . They us [et al.  in R. raet al. [VL can be diagnosed by cytologic or histopathologic identification of organisms, isolation of amastigotes, detection of serum antibodies, or PCR . Weese eet al.  recommenet al. ,15. The et al. ,22 usingR. norvegicus) under study. Milena et al. [R. rattus in Turkey, although rats harbored live parasites capable of infecting sand flies. Similarly, Ashford [R. norvegicus but maintained longer infectivity for sand fly vectors. In other studies [We did not observe any clinical signs of leishmaniasis in brown rats . In our study, we did not find statistically significant differences in the age and sex of rats. Further, research involving a higher number of rat samples might answer this difference in sex and age.Caldert et al.  reportedR. norvegicus) from Grenada. The seroprevalence of VL found in the present study suggests that rats are frequently exposed to the parasite in Grenada. Although there is no report of human leishmaniasis in Grenada, the presence of moderate prevalence of antibodies against Leishmania spp. in R. norvegicus on the island could suggest rats may play a role in the transmission of VL to humans. Rats live in close proximity to humans, and they could play a role in the epidemiology of the parasite in Grenada and the Caribbean nations. Therefore, regular active surveillance for Leishmania spp. in rats and phlebotomine sand flies in Grenada is warranted. The results show that rats (R. norvegicus) in Grenada are exposed to Leishmania spp. The rats could play an important role in the transmission of leishmaniasis to humans and other animals in Grenada.This is the first report of antibodies to VL in brown rats (RNS planning and overseeing of the research project and manuscript writing; KT helped in trapping the rats, anesthesia, and collection of blood; and ARD and NC performing ICT, interpretation of data, and review of the manuscript. All authors read and approved the final manuscript.