Cryptococcus neoformans were increasingly recognized as a significant threat to thehealth of immune compromised populations throughout the world. Until recently, the closely relatedspecies C. gattii was considered to be a low-level endemic pathogen that was confined totropical regions such as Australia. Since 1999, C. gattii has emerged in the Pacific Northwestregion of North America and has been responsible for a large disease epidemic among generallyhealthy individuals. The changing epidemiology of C. gattii infection is likely to be a consequence of alterations in fungal ecology and biology and illustrates its potential to cause serious human disease.This review summarizes selected biological and clinical aspects of C. gattii that areparticularly relevant to the recent North American outbreak and compares these to the Australian and SouthAmerican experience.During the latter half of the twentieth century, fungal pathogens such as Candida remain the most common cause of invasive yeast infection; however,opportunistic filamentous fungi such as Aspergillus spp, Fusarium spp, Scedosporium spp, Penicillium spp, and the zygomycota are becoming more prevalent in oncology and transplant centers [Cryptococcus neoformans increasedsignificantly since the onset of the HIV epidemic and continues to be commonamong individuals that do not have access to effective antiretroviral therapy [C. gattii, a closely related speciesthat was traditionally associated with tropical and subtropical climates, isnow gaining prominence as a cause of human and veterinary disease in NorthAmerica. While most clinical cases have occurred among generally healthyindividuals that reside in the Pacific Northwest, a few tourists or visitors tothe region have also been affected. The purpose of this review is to summarizeimportant biological and clinical characteristics of C. gattii that are relevant to the understanding of human diseasecaused by this emerging fungal pathogen.Although less than 500 of the estimated 1.5 million species of fungipose a threat to humans and animals , the pre centers , 4. Hum therapy . C. gattC. neoformans was first isolated in 1894 from fermented peach juice by the ItalianFrancesco Sanfelice [anfelice . Since tanfelice . Cryptoanfelice . Infectianfelice , 13.C. gattii was first isolated from a leukemic patient in 1970 and described asa variant of C. neoformans [C. gattii is closely related to C. neoformans, although its distribution is not global. C. gattii is typically restricted to tropical and subtropicalgeographical regions such as Australia, Brazil, and southern California [C. gattii belongs to serotypes B and C, while C. neoformans belongs to serotypes A and D [C. neoformans, C. gattii typically causes pneumonia andmeningitis [C. gattii appears to have a greater propensity to infect immune competent humans [oformans .C. gattlifornia , 16. In A and D . Like C.ningitis . Howevert humans , 19.C. gattii has acquired the ability to colonize new biogeoclimaticregions and is responsible for a recent outbreak of infection among humans andanimals in the temperate climate of Vancouver Island, British Columbia (BC),Canada [C. gattii infection were identified, including 8 fatalities [C. gattii infection on Vancouver Island peaked at 36 cases/millionpeople/year, a number that was significantly higher than the 0.94 cases/millionpeople/year observed in endemic regions of Australia [C. gattii infection on Vancouver Island was pneumonia [C. gattii infection on the Pacific coast of North America, it will be important forclinicians and laboratory scientists to remain vigilant for diseases that maybe caused by this fungal pathogen.For reasons that are not yet fully understood, ,Canada . Betweentalities . Betweenustralia , 23. Theneumonia . Althougneumonia , 24, 25.Cryptococcus is a largely polyphyletic genus that consists of at least 37different species and belongs to the kingdom Fungi, phylum Basidiomycota,class Tremellomycetes, and order Tremellales [C. neoformans species complex was firstclassified according to structural variations of the extracellularpolysaccharide capsule that are distinguished by agglutination assays withantigen-specific antibodies [C. neoformans wasclassified into four serotypes, A through D, in the 1950s and 1960s [C. neoformans species complex, and rarehybrid serotypes between C. neoformans and C. gattii such as BD and AB havealso been observed [mellales –28. Themellales , 27, 29.tibodies , 31. Usnd 1960s , 32. Theobserved –37.C. gattii was initially classified as a variety of C. neoformans, bearing the name C. neoformans var. gattii or var. bacillispora [C. gattii and C. neoformans were shown to havesubstantial differences in their biochemistry, ecology, epidemiology, andclinical manifestations  and C. gattii (serotypes B and C). The sexual state of C. gattii is known as Filobasidiella bacillispora [llispora , 38. Sue review ) as a ree review , 39. Fo complex , 41,cryC. neoformans may be classified into VNI/VNII genotypes corresponding toAFLP1/1A/1B (serotype A), VNIII/AFLP3 (serotype AD), and VNIV/AFLP2 (serotypeD), while C. gattii may bedistinguished as VGI/AFLP4 (serotype B), VGII/AFLP6 (serotype B), VGIII/AFLP5(serotype B or C), and VGIV/AFLP7 (serotype B or C). Interspecific hybridserotypes BD and AB correspond to AFLP8 and AFLP9, respectively [C. gattii isolates have aVGII genotype that is further subtyped into VGIIa (major) and VGIIb (minor) [C. gattii. Based on thesefindings, it was suggested that these lineages should be considered as separatetaxa, similar to the two monophyletic lineages within C. neoformans that correspond to varieties grubii and neoformans [C. gattii strains havecolonized a novel environment, they are not phylogenetically unique [The most recentclassification of cryptococci was established by genetic typing using PCRfingerprinting, random amplification of polymorphic DNA (RAPD), amplifiedfragment length polymorphism (AFLP) analysis and multilocus sequence typing(MLST) , 42–45. ectively , 37. The (minor) . A receny unique .C. gattii is endemic in tropical and subtropical regions such as Australiawhere it is most commonly associated with eucalyptus trees, particularly Eucalyptus camaldulensis and Eucalyptus tereticornis [Prunus dulcis), golden shower (Cassia fistula), and Douglas fir (Pseudotsuga menziesii) in Colombia, Brazil, and Vancouver Island,respectively [C. gattii has also been isolated frominsect frass in Australia and a wasp nest in Uruguay [Cryptococcus amylolentus, Tsuchiyaeawingfieldii, and Bullera dendrophila,have also been isolated from insect frass [C. gattii in the Pacific Northwest isintriguing, since this temperate climate is characterized by mild and wetwinters and warm and dry summers. On VancouverIsland, C. gattii has mainly beenfound on trees in the coastal Douglas fir (CDF) biogeoclimatic zone such asfir, cedar, and maple [ticornis , 47–49. ectively , 47. C.  Uruguay , 51. Otct frass –54. In tnd maple , 47. Thend maple , 23, 47.C. gattii. A survey conducted in 16countries revealed that the molecular types mostcommonly found in the clinic and environment were VGI and VGII [C. gattii clinicalinfection is rare in this continent, represented by only six of 535 serotyped Cryptococcus isolates [C. gattii isolatesidentified were not determined. In Australia, the C. gattii VGI genotype is the most common clinical or environmentalisolate, while the VGII genotype is an infrequent cause of human or animalinfection [C. gattii infection. Interestingly, most human cases in this region are due to VGII,suggesting that C. gattii hascolonized a new environmental niche [Ecological andgeographical differences exist among the different molecular types of and VGII . Genotypand VGII , 55–58. isolates . In thisnfection , 50. A nal niche , 61. Thial niche .C. gattii isolates in Australia's Northern Territory and on Vancouver Islandare of the VGII genotype, a notable difference between the two regions is theremarkably limited diversity of the latter. Specifically, ninety five percentof the environmental and clinical samples on Vancouver Island are of the VGIIgenotype, and within this group 90% are of the major genotype VGIIa [C. gattii strains: the 1975 Seattle human isolate NIH 444  and the 1992 San Francisco environmental isolate CBS 7750 isolatedfrom E. camaldulensis [C. gattii clinicaland environmental isolates from South America have shown that VGII predominatesover the other molecular types in both Colombia and Brazil as observed onVancouver Island [α mating-type cells within the Brazilian C. gattii VGII population and the fact that all Vancouver Island C. gattii isolates found to date havealso been of the α mating type [C. gattii VGII isolates (serotype B) in Colombia are mainly ofthe opposite a mating type, although large numbers are present in regions thathave a temperate climate that is similar to Vancouver Island [While the majority of pe VGIIa . MLST stdulensis . These sdulensis . These dulensis . Notablydulensis . Thereadulensis . The VGIdulensis , 46. Rer Island , 64, 65.ing type , 65, 66.C. gattii and C. neoformans share many attributes that increase their ability toinvade and survive in a host organism [C. gattii to date include an outer polysaccharide capsule, melanin,mannitol, extracellular proteinase, products of the laccase pathway, superoxidedismutase, phospholipases, urease, and the STE12α transcription factor (ahomologue of Saccharomyces cerevisiae STE12) that is present only in the α mating type  to mice suggests thaturease may aid in the dissemination of the fungus from the lung to the centralnervous system, although the exact mechanism is unknown [C. gattii was observed to share an endophytic relationship with decaying wood of botheucalypt and noneucalypt trees, where the laccase enzyme system appears toplay a role in digestion of lignin [A carbohydrate-richouter capsule that is composed primarily of glucuronoxylomannan (GXM) withsmaller proportions of galactoxylomannan  and mannoproteins is the majorvirulence factor for both oformans . The capoformans , 95. Kiognition . Speciesglobulin –82. Thismination . Similar. gattii . Intratr unknown . It is af lignin , 50.C. neoformans and C. gattii appear to share many of the same virulence factors, it is intriguingthat C. gattii most commonly infectsimmune competent individuals while C. neoformans primarily infects the immune compromised host. One explanationfor this observation is that immune compromised individuals simply have moreenvironmental exposure to C. neoformans compared to C. gattii [C. gattii disease [C. neoformans with comprehensive profiling ofindividual mutants in an animal model [C. gattiiand may in turn stimulate the development of novel therapeutic strategies.Giventhat . gattii . Further disease . Modern al model . A similC. gattii. The term phenotypicswitching refers to an adaptive mechanism characterized by structuralmodifications of the extracellular capsule and cell wall [C. neoformans [C. gattii strain NP1 has also been identified[C. gattii NP1 phenotypic variant demonstrated increased virulenceof the mucoid form. In this study, both mucoid and smooth variants were foundin the lung homogenates while only the smooth variant was found in the brainhomogenates. In terms of the immune response, infection with the smooth variantelicited a greater inflammatory response as characterized by lymphocyte andmonocyte infiltration and gave rise to smaller cryptococcomas than the mucoidvariant [Phenotypic switching andsame-sex mating are two interesting attributes that contribute to the virulenceof ell wall . This phoformans , 100. Rentified. Evaluatvariant .C. gattii isolates, it wasdemonstrated that all Vancouver Island isolates belong to the α mating-type andare unusually fertile [C. gattii undergoes same-sex mating, a process that has beenstudied in C. neoformans [α haploid cells can undergo sexualrecombination via fruiting, a process by which haploid cells fuse, chromosomalreassortment and recombination occur, followed by meiosis and sporulation [α cells (rather than an α and an a cell) may confera survival advantage and could explain why all Vancouver isolates are of the αmating type [C. neoformans have been observed in the laboratory but not innature [In contrast to mostAustralian  fertile , 66. Theoformans . Upon nurulation . Same-sing type . Both trnnature , 102.C. gattii infection have been reported in literature. One grouphas investigated the survival of A/J inbred mice following intranasal infectionwith different Vancouver Island C. gattii molecular types. In this report, the major genotype VGIIa appeared mostvirulent , the minor genotype VGIIb wasavirulent , and the VGI genotype wassimilar in virulence to VGIIa [A limited number of animal model studies of to VGIIa . These fto VGIIa , 24, 103C. gattii isolates that werenot obtained from Vancouver Island. One study compared mouse and humanpulmonary inflammatory responses to intratracheal infection with 10 differentAustralian isolates of C. gattii [C. gattii yeast, anddestruction of lung tissue. In humans, variablepulmonary pathology has been detected including poorly formed granulomascontaining intracellular C. gattii,lymphocytic interstitial pneumonitis, tissue necrosis, and fibrosis. In bothspecies the local lymphocyte pool consisted largely of T-cells with a 2:1 ratioof CD4 to CD8 cells in humans [Furtherinsights into disease pathogenesis have been derived through animal studiesusing . gattii . In BALBn humans .C. gattii 9714  under differentexperimental conditions [C. gattii disease,contrasting with the apparent predilection of immune competent humans to thisinfection. At high doses of C. gattii,immune suppressed mice were more likely to develop severe disease compared toimmune competent mice. SCID mice that lack T- and B-lymphocytes were moresusceptible to infection relative to wild-type BALB/c, suggesting a protectiverole for these cell types [C. neoformans 9759  has increased virulence compared to C. gattii 9714 after systemic infection of BALB/c mice, regardlessof the immune state of the animal. Specifically, C. neoformans 9759 infected the brain and lungs of BALB/c mice,while C. gattii 9714 affected thelungs and skin. Ulcerative lesions on the tail due to intravenous C. gattii infection were more common inimmune competent mice, while rectal prolapse was more common in immunesuppressed mice. This study also demonstrated that infection by either C. neoformans 9759 or C. gattii 9714 may causegastrointestinal pathology, although its incidence was rare [A second study examined the pathology seen in BALB/c mice followingsystemic infection with the clinical isolate nditions , 106. Itll types . Comparawas rare .Cryptococcus isolates administered to BALB/c mice via theintraperitoneal route [C. gattii GR52 and GR56 (both serotype B), from immune competent goats thatdied of pneumonia in Spain; C. gattii B4506(serotype B), an Australian environmental isolate known to be highly pathogenicin mice; C. gattii I-682 (serotypeC), from a Colombian native almond tree; and C. neoformans GR297 (serotype D), from an AIDS patient in Spainwith meningitis. Two of five mice infected with C. neoformans GR297 developed liver and peritoneal abscesses andone mouse died after four weeks. Fungal cultures of spleen, liver, kidney,testes, lung, and brain were done for each group and were positive in at leastone organ for GR52, GR297, GR56, B4506, and I-682 in 80%, 77%, 70%, 70%, and33% of the cases, respectively [Cryptococcus (91%), followed by the liver (75%), kidney (75%),testes (71%), lung (62%), and brain (29%). C. gattii GR52, the cause of a pneumonia outbreak in goats, was also isolatedfrom the lungs of all BALB/c mice studied, suggesting that it has a particulartropism for lung tissue. Pathologic findings associated with infection of thebrain included areas of spongiosis in the white matter with an abundance ofencapsulated yeasts in the basal ganglia while infected lungs showedencapsulated yeasts within alveoli, peribronchial vessels, and interalveolarspaces. Interestingly, inflammation was not observed in either organ amonginfected mice [A third study examined the pathogenesis of five al route . The isoectively . Based oted mice .C. gattii administration through thetail vein, and significant involvement of the visceral organs and peritoneum inthe third animal study may have resulted from intraperitoneal inoculation ofthe fungus. Nonetheless, the tissue tropism observed in humans is similar tothat seen in these mouse models [C. gattii isolate, infectious dose, and immune status of the host also play a role inthe virulence of the fungus and the severity of infection [These studies show that the route of experimental cryptococcalinfection is a major determinant of the site of disease. In general,intravenous infection directly induces systemic disease, whereas inhalation ofthe fungus via the airway mimics the natural route of infection and leads toprimary disease in the lung . For exae models , 15, 21.C. gattii has the potential to infect awide range of animals throughout the world, including wild, farm, domestic, andaquatic animals, along with a variety of birds. This broad host range wasobserved during the recent outbreak in British Columbia [C. gattii infection in domestic animals in southwestern BC,involving 78 feline and 51 canine cases [C. gattii infection,and that all cases in dogs and 50% of cases in cats were due to C. gattii serotype B [C. gattii disease differs slightly in cats and dogs. Among canines, theaffected organs included the respiratory system (52%), central nervous system(42%), and the skin or gastrointestinal tract (6%), while in felines 56% wererespiratory, 26% involved the central nervous system, and 19% were dermal withno cases of gastrointestinal involvement [Columbia , 110. InColumbia , 111–113ne cases . It was rotype B . The tisolvement .C. gattii. Of the 91 animalsthat underwent swabbing of the nasal cavity, two eastern grey squirrels werefound to be positive for C. gattii. This number is similar to the incidence of infection in asymptomatic companionanimals; specifically 1.1% of dogs, 4.3% of cats and 1.5% of horses werepositive for C. gattii [C. gattii infection in dogs and cats does not necessarily progress to clinical disease. In some cases it may be cleared while in other cases it may persist or developinto more severe disease [C. gattii from Vancouver Island to geographic regions that do not harbor theorganism and that veterinary cases may also be a reliable sentinel of humandisease [C. gattii infection was identified inanimals prior to the human outbreak that began on Vancouver Island in 1999 andthat nearly twice as many animals appeared to be infected with C. gattii compared to humans between1999 and 2003 [C. gattii has not been reported to date,transmission of C. neoformans frompet birds to humans has been documented, and possible human-to-humantransmission has also been described [C. gattii involving goats in Spain [Another study wascarried out on Vancouver Island to determine whether wild animals were infectedwith . gattii . Subclin disease . It has disease . The latand 2003 . These oescribed –119. Inin Spain  and psitin Spain  have alsC. gattii may cause mild to severe clinical disease in the apparently healthyas well as immune compromised host. Like other cryptococcal species, C. gattii enters the human host throughthe inhalation of airborne propagules and targets the lung as a primary site ofinfection [C. gattii infectioninvolve the lungs in the tropical climate of Australia (66% pulmonary) as wellas the temperate climate of Vancouver Island and its surroundings (75%pulmonary) [nfection , 108. Innfection , 15, 82.lmonary) , 60.C. gattii infection on VancouverIsland from 1999 to 2006 [C. gattii infection were pulmonary (75%), neurological (8%), combined (9%),and unknown (8%). Of those patients with combined disease, the main sitesinvolved the lung and CNS, and less commonly the skin and lung, or skin andCNS. Death was a rare outcome of C. gattii infection on Vancouver Island with 8 fatalities from 1999 to 2006,or approximately one death per year. Of the deceased patients, five presentedwith both pulmonary and CNS pathology, three had other underlyingcomorbidities, and one had an adverse reaction to therapy. The mean age of thedeceased patients was 61 years (range from 26 to 87) [A study performedby the British Columbia (BC) Cryptococcal Working Group at the University ofBritish Columbia reviewed the clinical aspects of 171 individual human cases of  to 2006 . It was  to 2006 . The cli6 to 87) .C. gattii on Vancouver Island or wereexposed many years earlier. This suggests that another source of C. gattii may exist outside of VancouverIsland, or that the fungus may have been transiently present in othergeographic regions due to dispersal mechanisms [C. gattii may remain latent in the bodyand reactivate at a later time [A summary of themost recent case reports associated with the Pacific west coast is presented inchanisms , 121. Ater time .C. gattii infection may bequite subtle. For example, patients 3 and 9 were asymptomatic while patient 6experienced only a nonspecific cough [In the BCCryptococcal Working Group study, the pulmonary symptoms most commonly observedin the 171 cases were cough and dyspnea, while the main symptom involving theCNS was headache . Similaric cough , 24, 114ic cough . The speic cough . Meanwhic cough , 25, 114C. gattii cases from the BC CryptococcalWorking Group study were caused by C. gattii serotype B [C. gattii serotypeC in the environment had not been described prior to 1998 [C. gattii majorgenotype VGIIa is highly associated with human disease: 82% of the cases wereVGIIa, while the others were VGIIb (9%) and VGI (6%) [C. gattii disease were smoking (50%), oral steroid use (30%), invasive cancer(24%), and chronic lung disease (13%). HIV infection (4%) and receipt of anorgan transplant (3%) were considered less common risk factors for thedevelopment of C. gattii disease inthis study population [All 171 rotype B . This fi to 1998 , 122. ItVGI (6%) . Similarpulation . ExaminaC. gattii on Vancouver Island has been estimated to be 6-7 months with a range from 2 to 11months [C. gattii infection. It is likely that the clinical incubation period depends on several variablessuch as differences in host immunity and the intensity of C. gattii exposure.Thus far, themedian incubation time of 1months . One exc1months . The potC. gattii infection and generally requires analysis of tissue or fluids from infectedsites such as cerebrospinal fluid, bronchial washings, blood, and urine [C. neoformans and C. gattii readilygrow as round cream-colored mucoid colonies on Sabouraud dextrose agar, aselective medium that is widely used for the isolation of yeast [C. neoformans or C. gattii fromother fungi [C. gattii colours the medium blue while C. neoformans does not (medium remains yellow) [Laboratory testingis essential to diagnose nd urine . Light mnd urine , 20, 91.nd urine . In the of yeast . Alternaof yeast , 47. Dier fungi . Furthe yellow) . Though  yellow) . Finally yellow) , 25.C. gattii to antifungaltherapy has been attributed to serotype-specific differences in antifungalactivity though there are no consistent or predictable data to support thishypothesis [C. neoformans and 42 C. gattii isolates tovarious antifungal drugs including amphotericin B, flucytosine, fluconazole,posaconazole, voriconazole, and isavuconazole [C. gattii serotypesB or C compared to other serotypes. In vitro activity of antifungal agentsmay be helpful in guiding the clinical management of severe cryptococcalinfection including meningoencephalitis . A slowepothesis , 128. Toconazole . The majC. gattii on Vancouver Island, andpossibly other adjacent regions, indicates that this fungus has the ability toadapt to new environmental conditions. It has been suggested by Kidd et al. that the temperate climate ofVancouver Island may provide a favorable niche for the survival and dispersalof C. gattii [C. gattii colonization of new geographicregions [C. gattii havebeen studied on Vancouver Island. Some trees in the region show intermittentpositivity, suggesting the movement of fungal spores [C. gattii levelsexist in public areas such as park entrances, parking lots, and beaches despitethe fact that these areas are characterized by low tree density [C. gattii dispersal appears to be mediated by human activity, including contact withfootwear and car wheel wells, deforestation, and gardening [The colonization of . gattii . Global regions . Studiesregions , 130. Thl spores . High C. density , 121. Aardening , may alsC. gattii colonized other regions on thePacific west coast? It is known that C. gattii (serotype C) was isolated from a eucalyptus tree in San Francisco in1992, but since then C. gattii hasnot been found in that environment. In order to determine whether C. gattii has spread to the UnitedStates, Fraser et al. investigatedpossible colonization on the San Juan Islands, the closest U.S. region toVancouver Island [Basidiomycota,namely Cryptococcus laurentii and Cryptococcus cellulolyticus, were isolated, C. gattii was not detected. This suggests that C. gattii may not have yet colonized the San Juan Islands, ordispersal of the fungus is impeded by a geographical barrier that is the Juande Fuca strait [C. gattii wasisolated from environments outside of Vancouver Island including the BritishColumbia mainland, the Gulf islands, and Washington state, but no positiveenvironmental samples were found in Oregon [C. gattii has beenisolated in Seattle (1975),Vancouver Island, and the British Columbia mainland, and most recently inOregon, Washington, and Alberta [Has r Island . The Sana strait . In a ren Oregon . Inthe  Alberta –25, 63.C. gattii infection in the Pacific Northwest highlights the fact that this fungusis an important emerging pathogen that can adapt to new potential environmentalniches. C. gattii isolates found onVancouver Island appear to be hypervirulent, as the number of infectedindividuals per million people per year between 2002 and 2005 was more than 36times higher on Vancouver Island compared to endemic regions such as Australia [C. gattii disease in order to discover effective prevention and treatment strategies,including measures to limit human exposure to this pathogenic fungus. Employing C. neoformans as a model organism tounderstand the disease-causing potential of C. gattii is no longer sufficient since fundamental disparities such asdifferences in host tropism and the expression of certain virulence factorsexist between the two species.The recent outbreak of ustralia , 23. It


Phyllanthus reticulatus on lipid profile and oxidative stress in hypercholesterolemic albino rats.This study was designed to investigate the effect of P. reticulatus treated (250 and 500 mg/kg body weight doses for 45 days). After the treatment period of 45th day triglyceride, VLDL-cholesterol, HDL-cholesterol, total cholesterol (TC), LDL-cholesterol and oxidative stress (protein carbonyl) were assayed and compared with hypercholesterolemic control.Hypercholesterolemia was induced in albino rats by administration of atherogenic diet for 2 weeks. Experimental rats were divided into different groups: normal, hypercholesterolemic control and P. reticulatus (250 mg and 500 mg/kg) produced significant reduction (P < 0.05) in triglyceride, VLDL-cholesterol, total cholesterol (TC), LDL-cholesterol and oxidative stress (protein carbonyl) while increased HDL-cholesterol in atherogenic diet-induced hypercholesterolemic rats at the end of the treatment period (45 days). However, the reduction in the above parameters was comparable with hypercholesterolemic control. Thus, aqueous extract of P. reticulatus is effective in controlling TC, lipid profile and oxidative stress in hypercholesterolemic animals.The aqueous extract of P. reticulatus can be utilized for prevention of atherosclerosis in hypercholesterolemic patients.The results suggest the aqueous extract of  Hypercholesterolemia is associated with serious health risks and increased mortality. Hypertension, hyperlipidemia, insulin resistance and glucose intolerance are known as cardiac risk factors that cluster in obese individuals. The pers2In the recent years, there is growing interest in herbal medicines all over the world. Traditional medicinal plants having antilipidemic property can prove to be an useful source for the development of new oral hypolipidemic agents or simple dietary adjuvant to existing therapies. Ethnopharmacological surveys indicate that more than 1200 plants are used in traditional medicine for their hypolipidemic activity.5 The hyp68Phyllanthus reticulatus (Family—Euphorbiaceae) is a climbing shrub which grows all over India.[Phyllanthus niruri has been shown to possess significant hepatoprotective activity in fatal liver diseases such as liver cirrhosis and hepatocellular carcinoma.[P. reticulatus has not been studied for significant chemical as well as biological studies, the plants of this genus were reported to contain lignins, flavonoids, triterpenoids, alkaloids and polyphenolic compounds.[P. reticulatus in rat model.er India. It has ber India. Phyllantarcinoma. Chemicalarcinoma. Althoughompounds. Flavonoiompounds. The presad libitum. The animals were maintained as per the CPCSEA guidelines and regulations and the study was approved by the Institutional Animals Ethics Committee at Bharathiar University, Coimbatore, India. The composition of atherogenic diet used during the study was given as in Adult albino rats (Wistar strain) weighing 180–200 g of either sex were used for this study. The animals were housed in polypropylene cages at controlled temperature (26 ± 2°C), relative humidity (60 ± 5%) with a 12–12 h light–dark cycle. The rats were fed with standard laboratory diet and water was provided P. reticulatus were collected in July–September from Maruthamalai Hill, Coimbatore (India). The plant was identified and authenticated with the help of Botanical Survey of India, Coimbatore (India). The plant material was dried under shade and powdered in a grinder. The powdered material (100 g) was extracted with double-distilled water by the hot continuous percolation method in a Soxhlet apparatus. The extract was evaporated to dryness under vacuum and dried in a vacuum desiccator to obtain a residue of 15.65 g.Aerial parts (stem and leaves) of et al. was followed.[In order to induce hypercholesterolemia, the method reported by Bopanna followed. The animGroup I: Normal diet.Group II: Atherogenic diet containing 1% cholesterol.P. reticulatus (250 mg/kg b.w.).Croup III: Atherogenic diet + aqueous extract of P. reticulatus (500 mg/kg b.w.).Group IV: Atherogenic diet + aqueous extract of At the end of the treatment, the rats were fasted overnight and killed by decapitation. Blood was collected and serum was separated and stored in a refrigerator (4°C) until assay.Total cholesterol (TC), serum trAll the chemicals were used of analytical grade, obtained from M/s. SISCO Research Laboratories Pvt. Ltd, Mumbai.t-test.[Statistical analysis was carried out using Student’s t-test.P. reticulatus. Feeding of atherogenic diet increased serum cholesterol, triglyceride, LDL-cholesterol and HDL-cholesterol level, VLDL-cholesterol and protein carbonyl level when compared to normal group at over a period of 45 days. Administration of aqueous extract of P. reticulatus (250 and 500 mg/kg per day) showed statistically significant decrease in total cholesterol (P < 0.05), triglyceride (P < 0.001), LDL-cholesterol (P < 0.05), VLDL-cholesterol (P < 0.001) and protein carbonyl level (P < 0.05) while increase in HDL-cholesterol level (P < 0.05) as compared to hypercholesterolemic animals [Phytochemical screening revealed the presence of lignins, flavonoids, triterpenoids, alkaloids, polyphenolic compounds and mucilage in the aqueous extract of  animals . The aqu animals .P. reticulatus on lipid profile and oxidative stress in hypercholesterolemic albino rats was evaluated in this study. P. reticulatus, an Indian herbal plant, possesses cardioprotective and lipid lowering properties. Treatment with P. reticulatus extract produced a significant decrease in the serum lipid level in atherogenic diet-induced hypercholesterolemia in rats. Maruthappan and Sakthi Shree[Adenanthera pavonina and Terminalia chebula lowered the total cholesterol and its fractions in lipoproteins. They reported hypolipidemic activity of the saponins, flavonoids and polyphenolic compounds from A. pavonina. Beta-sitosterol, a phytosterol, is reported as useful in the treatment of hyperlipidemia.[et al.[Solanum melongena are reduced in hyperlipidemia. The aqueous extract of P. reticulatus contains lignins, flavonoids, triterpenoids, alkaloids, polyphenolic compounds and mucilage. The high amount of lignins, flavonoids present in P. reticulatus may be responsible for the hypocholesterolemic effect. Use of diet rich in saturated fats and an increase in coronary heart disorder (CHD) has been observed in the developing countries for the past few decades.[The effect of thi Shree23 found ipidemia. Sudhessha.[et al. reported decades.P. reticulatus reduced the atherogenic diet-induced hypocholesterol manifestations in multiple ways. Increase in the level of serum triglyceride, total cholesterol, LDL-cholesterol and VLDL-cholesterol in the P. reticulatus-treated group indicate that phytochemicals may be interfering with metabolism or biosynthesis of lipids. Pretreatment with P. reticulatus showed reduction in serum lipid levels with concomitant increase in HDL-cholesterol. Decrease in serum lipid profiles and increase in HDL-cholesterol in P. reticulatus-treated group may be due to the present of phytochemicals. Lipid lowering effect of P. reticulatus could be due to inhibition of hepatic cholesterol biosynthesis, increased fecal bile acid secretion and stimulation of receptor-mediated catabolism of LDL-cholesterol and increase in uptake of LDL from blood by liver.[P. reticulatus extract, indicating the beneficial effect of extract in cardiovascular diseases.Pretreatment of by liver. The atheby liver.28 were sOxidative stress is an important event in the development and maintenance of atherosclerosis. Protein P. reticulatus. Aqueous extract of P. reticulatus can be utilized for prevention of atherosclerosis in hypercholesterolemic patients.In this study, an increase in serum HDL-cholesterol with a concomitant decrease in other lipids was observed. It can be concluded from the present data that the levels of total cholesterol, triglyceride, LDL-cholesterol, VLDL-cholesterol and protein carbonyl which are raised in atherogenic diet are lowered significantly with the aqueous extract of


Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae. The development of a high-throughput molecular species diagnostic assay now enables routine PCR-based surveillance of malaria infections in large field and intervention studies, and improves resolution of species distribution within and between communities.When diagnosed by standard light microscopy (LM), malaria prevalence can vary significantly between sites, even at local scale, and mixed species infections are consistently less common than expect in areas co-endemic for This study reports differences in the prevalence of infections with all four human malarial species and of mixed infections as diagnosed by LM and post-PCR ligase detection reaction – fluorescent microsphere (LDR-FMA) assay in 15 villages in the central Sepik area of Papua New Guinea.P. falciparum, P. vivax, P. malariae and Plasmodium ovale were observed in LDR-FMA compared to LM diagnosis (p < 0.001). Increases were particularly pronounced for P. malariae (3.9% vs 13.4%) and P. ovale (0.0% vs 4.8%). In contrast to LM diagnosis, which suggested a significant deficit of mixed species infections, a significant excess of mixed infections over expectation was detected by LDR-FMA (p < 0.001). Age of peak prevalence shifted to older age groups in LDR-FMA diagnosed infections for P. falciparum  and P. vivax  but not P. malariae infections . Significant geographical variation in prevalence was found for all species , with the extent of this variation greater in LDR-FMA than LM diagnosed infections . Insecticide-treated bednet (ITN) coverage was also the dominant factor linked to geographical differences in Plasmodium species infection prevalence explaining between 60.6% – 74.5% of this variation for LDR-FMA and 81.8% – 90.0% for LM , respectively.Significantly higher rates of infection by The present study demonstrates that application of molecular diagnosis reveals patterns of malaria risk that are significantly different from those obtained by standard LM. Results provide insight relevant to design of malaria control and eradication strategies. Plasmodium species prevalence and determine the causes of the often pronounced local heterogeneity in malarial prevalence [Malaria is an infectious disease that shows considerable spatial heterogeneity on global ,2, regioevalence .Plasmodium species share both host and vector, mixed species infections are commonly observed. An extensive review of the literature on mixed species malaria infections by Ritchie [Plasmodium falciparum, Plasmodium malariae and Plasmodium vivax mix, a deficit of mixed infections has generally been recorded [P. falciparum, P. malariae and Plasmodium ovale [Plasmodium species.As all human  Ritchie  concluderecorded ,15, suggum ovale ,15. Howeum ovale . More reum ovale  and randum ovale ,19. EarlP. falciparum, P. vivax, P. malariae and P. ovale), and mixed species infections are very common [P. malariae and P. ovale infections [Previous studies on spatial patterns of malarial infections were based exclusively on light microscopy diagnosis (LM) of infection, which has a significantly lower sensitivity for detecting malarial infections compared to PCR-based diagnostic assays ,20. The y common . In thesfections  as well fections .P. falciparum, P. vivax, P. malariae and P. ovale simultaneously and is performed in 96-well plate format to ensure efficient sample processing. Overall high specificity and sensitivity of the assay provides more accurate assessment of minority species in mixed infections. In addition, good correlations between parasite density and median fluorescence intensity confirmed that the assay is semi-quantitative [falciparum compared to falciparum infections, and a significant shift in distribution of infections to older age groups by LDR-FMA compared to LM diagnosis was observed [P. vivax compared to P. falciparum blood stage infections when diagnosed by LM but identical rates when PCR diagnosis was used [The developments of a high-throughput post-PCR, LDR-FMA species typing assay  now maketitative ,24. In awas used .Plasmodium species. In order to further evaluate the effect of the increased sensitivity of detection on patterns of Plasmodium species infection, here LM and LDR-FMA diagnostic methods were compared in a series of 15 cross-sectional population surveys at five distinct locations in the central Sepik region of East Sepik province, PNG.It is, therefore, becoming clear that increased application of PCR-diagnosis significantly alters the understanding of the epidemiology of different 2 that is characterized by low hills, plains and riverine plains with a wet tropical climate [P. falciparum, 12.1 for P. vivax, and 9.6 for P. malariae [P. falciparum to be significantly reduced [The selected study sites in the Maprik and Wosera-Gawi districts cover a combined area of over 160 km climate . The nat climate  is situa climate , with enmalariae . By comp reduced .For the present study five distinct areas surrounding local health facilities  were selected Figure . BrukhamFrom each household a semi-structured questionnaire was administered to collect data on type of house, household assets, education of the parents, personal use of bednets, recent health facility attendance and use of antimalarial drugs. From each individual, a thick and thin film was prepared on a single slide and a 250 μl blood sample collected into a K+EDTA microtainer from a finger or heel prick. Haemoglobin measurements were made from this blood sample using a HemoCue 201+ Hb meter ; remaining blood was preserved for extraction of DNA.A socio-economic index was created using data on household ownership of consumer durables . Households that owned < five consumer durables were classified as "low", > five consumer durables were classified as "medium", those with > 10 as "high" socio-economic status.P. falciparum only. All slides with densities less than 200/μl, along with a randomly selected 20% of all blood films were routinely re-examined. If less than 80% concordance was achieved between LM evaluations, the entire batch of slides was re-read.All smears were stained with 2.5% buffered Giemsa (pH 7.2) for 35 minutes and examined by LM. Slides were declared negative if no parasites were seen in 100 thick film fields. The parasite species in positive films were identified and densities were recorded as the number of parasites/200 WBC. Densities were calculated assuming 8,000 WBC/μl . Sexual Parasite DNA was extracted from the cell pellets using QIAmp 96 DNA Blood kits .P. falciparum, P. vivax, P. malariae and P. ovale. The design and sensitivity of this assay has been described previously [® FlexMAP™ "classification" bead sets (5'), and receive "reporter" labelling following incubation with streptavidin-R-phycoerythrin that binds to biotin (3'). Doubly labelled species-specific LDR complexes are detected using a Bio-Plex array reader . Species-specific fluorescence data were collected by Bio-Rad software, Bio-Plex Manager 3.0 . In order to guarantee maximum sensitivity for the detection of Plasmodium infections the PCR-cycle number was set at 35. Differentiation of negative from positive fluorescent signals was performed by comparing median fluorescent intensity (MFI) from study participants against values obtained from two negative controls on each 96-well plate. Cut-off values for positivity were set at the two standard deviations above the mean MFI of negative controls.A semi-quantitative post-PCR, ligase detection reaction/fluorescent microsphere assay (LDR-FMA)  was usedeviously ,24,28. I2 test, multivariate predictors of infection risk were determined using logistic regression. Non-parametric Spearman's correlations were used to compare parasite density by light microscopy and mean fluorescence intensities, while parametric correlations were calculated for the association of prevalence of infection and mean haemoglobin at village level.Differences in prevalence of malarial infections among villages and areas and univariate association between categorical explanatory variables were tested using χWithin and between village variance in risk of malarial infections were estimated using a hierarchical Bayesian logistic model, i.e.i ~ Bernoulli(pi)ri) = βa(i) + σj(i) + θj(i)k(i).where logit " is a fixed effect for the age group, "σj(i)" is a random effect for the area and the "θj(i)k(i)" is a random effect term for the village. In the models adjusting for bednet coverage, an additional linear effect is introduced into the model. All models were fitted using WinBugs 1.4 [and "i" indexes the individuals, "a" the age-groups, "j" the area and "k" the village within area. "rBugs 1.4  assumingj(i)/(σj(i)+ θj(i)k(i)). The effect of difference in bednet coverage on spatially structured variance was determined by comparing the individual variance component from models with and without inclusion of the bednet covariate. Variance components, their relative sizes, and changes following adjustment for bednet coverage are all reported on a logit scale.Variance components were estimated for all species with and without adjusting for bednet coverage. The relative contribution of between-area variance was determined by dividing the area variance component with the total spatially structure variance, i.e σA total of 2,744 volunteers from 15 villages in five distinct geographical areas  for all species except P. ovale which is rarely detected by LM in PNG [P. falciparum: 667, P. vivax 561, P. malariae 251, P. ovale 121), very few LM-positive infections were not detected by the LDR-FMA assay . Fluorescence intensities of samples that were positive by both LM and LDR-FMA were significantly higher than those that were only LDR-FMA positive . For infections positive by both methods, fluorescence intensities were significantly correlated with densities by LM for P. falciparum  and P. vivax  but not for P. malariae .In this population, 1,121 (44.4%) and 1,844 (73.0%) participants had LM and LDR-FMA detectable M in PNG . While tP. falciparum was the most common parasite with a prevalence of 29.9%  followed by P. vivax and P. malariae with a prevalence of 14.6%  and 3.9% , respectively  respectively, followed by adolescents , 2–3 year olds , adults ) and infants . P. vivax reached peak prevalence in younger children  than P. falciparum  or P. malariae .By LM, P. falciparum prevalence increased 1.8 fold , P. vivax 2.4-fold  and P. malariae 3.4-fold , while P. ovale was detected in samples from 4.8% (p < 0.001) of all participants. In parallel with these increases from LM to LDR-FMA, more mixed infections were detected . Fewer mixed species infections were observed in Burui and Wombisa compared to the other areas  in all species except P. vivax, where infections were most commonly found in children 7–9 years of age .For all n Figure . As a con Figure . PrevaleP. vivax a decrease was only observed at high ITN coverage (> 90%). People living in households where the mother spoke English or had attended at least 3 years of schooling also had a significantly lower risk of being infected with P. falciparum, P. vivax (LDR-FMA diagnosed only) and P. malariae infections but not with P. ovale. Only risk of infection with P. falciparum was significantly reduced in households of higher socio-economic status, while difference in house construction was only associated with difference in risk of LDR-FMA diagnosed P. falciparum and P. malariae infection.The risk of infections with malaria parasites was independently associated with a number of risk factors. The most important predictors were coverage of insecticide treated ITNs, educational level of the female household head and socio-economic status of family Table . ITN covPlasmodium species infections among villages  p = 0.03), and areas  p = 0.06) and P. ovale (LDR-FMA) p = 0.01). By LM, infection prevalence for individual villages varied between 15.5% to 45.2% for P. falciparum (coefficient of variance (CV): 26.4%), 6.4% to 25.1% for P. vivax (CV: 39.8%), 0.6% to 15.4% for P. malariae (CV: 94.5%) and 32.0% to 67.6% for any Plasmodium infections (CV: 21.9%). P. vivax and P. malariae infections were found more commonly in the villages in the foot hills (Ulupu (P. vivax only), Brukham & Ilaita) compared to the villages situated on the Sepik River flood plain. The prevalence of LDR-FMA detectable infections were significantly associated with those by LM . Although variation in infection prevalence between villages was less pronounced ; P. vivax: 22.9% to 45.2%, CV: 16.8%; P. malariae: 2.5% to 30.9%, CV: 55.3%); P. ovale: 1.2% to 9.0%, CV: 45.3% and any infection: 59.1% to 85.6%, CV: 17.8%), broadly similar geographical trends were observed. Interestingly, prevalence of any malaria infection by both LM and LDR-FMA was significantly associated with a lower population mean Hb level  and a higher incidence of moderate-to-severe anaemia . When diagnosed by LM, the reductions in age- and gender-adjusted Hb levels were most strongly associated with the presence of a mixed infection , followed by P. malariae , P. falciparum  and P. vivax single infections . Among LDR-FMA diagnosed infections only mixed species  and P. falciparum single infections  were associated with a significant reduction in Hb levels. Hb reductions were larger in children than adults and adolescents.For all species and both diagnostic methods, there were significant differences in prevalence of Plasmodium species and between methods of diagnosis  as well as P. vivax  and P. malariae  but not P. falciparum (r2: -0.15) when diagnosed by LM. Accounting for differences in bednet coverage among villages consequently reduced total spatial variance by 56–85% for all species and both diagnostic methods, except for LM-diagnosed P. falciparum infections (-2%). In contrast, substantial differences were observed between the two diagnostic methods and between Plasmodium species in regard to the spatial level at which the effect of bednet coverage was most pronounced  compared to LDR-FMA diagnosed infection (34–90%).The prevalence of e Figure . Such a P. malariae and P. ovale infections.With over 2,500 samples processed, the present study is the largest epidemiological study to-date that has used a post-PCR LDR-FMA assay for the diagnosis of malarial infection on all samples collected. The direct comparison of LM and LDR-FMA results confirmed that the LDR-FMA assay is both significantly more sensitive than LM and semi-quantitative ,24, withet al. [The higher sensitivity of the LDR-FMA in detecting low density infection was most notable in the assessment of mixed species assemblages and in age-specific prevalence. In concordance with the observations by Kasehagen et al. , a signiet al. . Having Plasmodium species have long been a subject of investigation, but results have been inconsistent and inconclusive . Since y Richie  found a sitaemia . A recensitaemia  that usesitaemia  that parPlasmodium species. While prevalence by LM and LDR-FMA was strongly correlated and overall geographical patterns comparable, LM-based prevalence showed a much higher degree of variability within individual areas for all Plasmodium species than LDR-FMA-based prevalence. In particular, for the non-falciparum species, almost all spatial variation in LDR-FMA-based rates of infection was observed as difference between areas. The proportionally higher within area variance is likely to be related to the lower sensitivity of LM (both for low-level infection and correct identification of infecting species). Differences in the quality of different slide batches, or between-reader variation may further contribute to within-area variation. In intervention trials where more than one slide is made and all slides have at least two independent reads these types of variability may be reduced. In large scale epidemiology studies or in monitoring of malaria interventions where thousands of people are screened, this is not necessarily common practice. If the trend for a decrease in survey-to-survey variation in PCR-based diagnosis holds true elsewhere, the heterogeneity of malaria risk at the local level may have been overestimated in earlier studies. Results of this nature would emphasize the importance of implementing molecular diagnostic methods.With both diagnostic methods considerable variation in prevalence of infections was observed among villages for all P. falciparum. Whether this reflects a true difference in ecology of different Plasmodium species in the study area is difficult to determine from a single cross-sectional survey. In addition, multiple P. falciparum strain infections are commonly observed in PNG [Interestingly, the least amount of between area variation (by both LM and LDR-FMA) was observed in the most common parasites, i.e. d in PNG  and the P. falciparium is well described [P. vivax, although the effect may be smaller than that against P. falciparum [P. malariae and P. ovale. While ITNs were highly effect in reducing P. malariae in a study in Burkina Faso[P. ovale infections.The coverage of ITNs in a given village was the best predictor of risk of malaria infections at individual and village levels. Both high coverage of ITNs in the village of residence as well as personal ITN use significantly reduced a participant's risk of being infected with any of the human malaria species. Interestingly, personal use was less strongly associated with protection (data not shown) than overall coverage in a given village. This suggests that if high coverage is achieved, ITN may have an effect on overall transmission and thereby also protect people who do not always sleep under a ITN . The proescribed  and therlciparum -37. To dkina Faso, this maP. falciparum, P. vivax and P. malariae in earlier studies in the Wosera [P. falciparum transmission by up to 90% [P. malariae and P. ovale prevalence suggest that ITNs may be quite effective for the control of these species, while control of P. vivax may be more difficult as indicated by the smallest reduction in spatial variation (by LDR-FMA). Interestingly, the variation in prevalence of infection was closely linked to a difference in mean haemoglobin levels in the study villages, indicating that control of malaria through ITNs may also result in improved haemoglobin levels. Given the well known problems with spurious correlations in ecological comparisons, these associations with ITN coverage need to be interpreted with care. Direct evaluation of the ongoing PNG national bednet distribution program on the transmission of non-falciparum species is needed.Differences in ITN coverage among villages also reduced total spatial variance of Plasmodium infections  by up to 85%. Similar spatial associations of coverage in untreated bednets with reduced (peak) prevalence and increased age at peak prevalence were observed e Wosera . In addie Wosera . In Afrip to 90% ,40. TherPlasmodium infections is the level of maternal education. It is well know that educated mothers have a better understanding of health, seek treatment more promptly, and use ITN more regularly [Plasmodium species infection.The only other factor consistently associated with a reduced prevalence of egularly -43 than egularly ,45. Simiegularly  may furtPlasmodium species infections. In combination with high-throughput Plasmodium strain typing assays [Plasmodium infections in the field and the understanding of malaria epidemiology is significantly improved., Future large scale malaria field studies and, in particular, intervention trials would therefore benefit significantly by improving capacity for molecular diagnosis of Plasmodium infections. Increased investment into establishing molecular diagnosis capacity in malaria endemic countries should be considered a high priority.The present study demonstrates that application of the high-throughput, PCR-based LDR-FMA diagnostic assay is feasible in large malaria field studies. Additionally, results show that epidemiological patterns of malaria risk detected by molecular diagnosis differ significantly from risk assessed by LM. Overall, results from this study enhance our understanding of the impact that low level asymptomatic infection, infection in older age groups, and infection by less common malaria parasites contributes to the burden and transmission of g assays ,48 and ng assays , the accThe authors declare that they have no competing interests.IM designed the study, analysed the data and wrote the first draft. SW, DM, SM, BK AS conducted the field work and read blood slides. SW and DM also assisted in data analyses. DTM and PAZ conducted LDR-FMA typing of all samples. TAS assisted with the spatial data analyses. All authors contributed to the interpretation of the data and the writing of manuscript.Table S1. Comparison of observed species mixtures in different regions by LDR-FMA.Click here for fileTable S2. Comparison of observed species mixtures in different regions by light microscopy.Click here for file


Panicum streak virus  is a close relative of Maize streak virus (MSV), the most serious viral threat to maize production in Africa. PanSV and MSV have the same leafhopper vector species, largely overlapping natural host ranges and similar geographical distributions across Africa and its associated Indian Ocean Islands. Unlike MSV, however, PanSV has no known economic relevance.Here we report on 16 new PanSV full genome sequences sampled throughout Africa and use these together with others in public databases to reveal that PanSV and MSV populations in general share very similar patterns of genetic exchange and geographically structured diversity. A potentially important difference between the species, however, is that the movement of MSV strains throughout Africa is apparently less constrained than that of PanSV strains. Interestingly the MSV-A strain which causes maize streak disease is apparently the most mobile of all the PanSV and MSV strains investigated.We therefore hypothesize that the generally increased mobility of MSV relative to other closely related species such as PanSV, may have been an important evolutionary step in the eventual emergence of MSV-A as a serious agricultural pathogen.GQ415386-GQ415401The GenBank accession numbers for the sequences reported in this paper are  Panicum streak virus (PanSV) is one of seven known African streak virus species within the Mastrevirus genus of the Geminiviridae. The best studied and most economically relevant species amongst the African streak viruses is Maize streak virus (MSV) which seriously constrains maize production throughout most of sub-Saharan Africa et al http://daEragrostis streak virus; cp: coat protein gene; LIR: long intergenic region; mp: movement protein gene; MSV: Maize streak virus; PanSV: Panicum streak virus; rep: replication associate protein gene; repA: RepA gene; SIR: short intergenic region; SSEV: Sugarcane streak Egypt virus; SSRV: Sugarcane streak Reunion virus; SSV: Sugarcane streak virus; USV: Urochloa streak virus.sacSV.ESV: The authors declare that they have no competing interests.AV, DPM, ALM, JML, SO, JZ, EKK, DPL, NM. JM, SS PGM and RWB collected isolates. AV, ALM, and LD cloned and sequenced the viruses. AV, PL and DPM conceived the project, AV and DPM analysed the data. AV and DPM prepared the manuscript and AV, PL, JML and DPM secured funding for the project's execution. EPR, PL and RWB provided ideas and comments during manuscript preparation. All authors other than PGM read and approved the final manuscript.Graph rationalizing the use with PanSV of the same 93% sequence identity strain demarcation threshold used for MSV. Graph rationalizing the use with PanSV of the same 93% sequence identity strain demarcation threshold used for MSV. The red and blue splines respectively plots the frequencies of pairwise sequence identities shared amongst 23 PanSV (253 pairwise distances) and 99 MSV isolates  at a resolution of 1% identity. Identity values were calculated with pairwise exclusion of alignment gaps .Click here for fileFull genome sequence alignments of 23 PanSV isolates. Annotated full genome sequence alignments of 23 PanSV isolates. Sequences either known or believed to have some role in mastrevirus replication and transcription are marked together with a corresponding label on the nucleotide sequence alignments. To highlight differences between the sequences, wherever nucleotides in a particular alignment column are identical to that of PanSV-A [ZM-Nya-g180-2007] they are replaced with a "-" character. In columns where they differ from PanSV-A [ZM-Nya-g180-2007] they are shown in lower case. "." characters indicate where gaps were inserted to align the sequences. [et al., 1991. Proc. Natl. Acad. Sci. USA 88:8029; [et al. 1985. Nucl. Acids Res. 13:4645; [et al. 1995. Virology. 208:303; [et al. 1984. Nucleic Acids Res. 13:7237; [et al. 1989. J. Gen. Virol. 70:2309; [et al.1997 Plant J. 12:1285; [et al. 1984. EMBO J. 3:3069; [et al.1991. Nucl. Acids Res. 19:4075; [et al. 1990. Plant. Mol. Biol. 15:865. ZM-Nya-g80-2007 t19:4075;  Fenoll eClick here for fileAnnotated predicted movement protein amino acid sequence alignments. Annotated predicted movement protein amino acid sequence alignments of 23 PanSV isolates. The hydrophobic, potentially membrane spanning internal domain of the sequences is highlighted. [lighted.  Wright eClick here for fileAnnotated predicted coat protein amino acid sequence alignments. Annotated predicted coat protein amino acid sequence alignments of 23 PanSV isolates. The potential nuclear localization signal and DNA binding domains  are highlighted on the sequence. [equence.  Liu et aClick here for fileAnnotated predicted replication-associated protein amino acid sequence alignments. Annotated predicted replication-associated protein amino acid sequence alignments of 23 PanSV isolates. Potential rolling-circle replication motifs and interaction domains inferred by analogy with MSV and Wheat dwarf virus are highlighted. [lighted.  Koonin &lighted.  GorbalenClick here for fileAnnotated predicted RepA amino acid sequence alignments. Annotated predicted RepA amino acid sequence alignments of 23 PanSV isolates. Potential rolling circle replication motifs and interaction domains inferred by analogy with MSV and Wheat dwarf virus are highlighted. [lighted.  Koonin &lighted.  Xie et aClick here for fileRecombination in PanSV. Details of the PanSV recombination events detected in this study including approximate breakpoint positions, parental-like sequences, and p-values for various recombination detection tests.Click here for file


Francisella (F.) tularensis is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, F. tularensis was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen  have been developed but are restricted to reference laboratories.F. philomiragia and all subspecies of F. tularensis were sequenced. Single nucleotide polymorphisms on species and subspecies level were confirmed by partial amplification and sequencing of 24 additional strains. Fluorescent In Situ Hybridization (FISH) assays were established using species- and subspecies-specific probes.The complete 23S rRNA genes of representative strains of F. philomiragia strains, and more than 40 F. tularensis strains tested. By combination of different probes, it was possible to differentiate the F. tularensis subspecies holarctica, tularensis, mediasiatica and novicida. No cross reactivity with strains of 71 clinically relevant bacterial species was observed. FISH was also successfully applied to detect different F. tularensis strains in infected cells or tissue samples. In blood culture systems spiked with F. tularensis, bacterial cells of different subspecies could be separated within single samples.Different FISH protocols allowed the positive identification of all 4 F. tularensis isolates from both laboratory cultures and clinical samples.We could show that FISH targeting the 23S rRNA gene is a rapid and versatile method for the identification and differentiation of  F. tularensis. This bacterial disease occurs in various clinical forms depending on the route of inoculation and the virulence of the F. tularensis strain involved [F. tularensis was long regarded to be restricted to the Northern Hemisphere [F. tularensis-like strains have been cultured in Queensland, Australia [F. tularensis has a broad host range and can affect more animal species than any other zoonotic pathogen [F. tularensis is often associated with water and aquatic fauna and its transmission is considered to be more complex involving blood-sucking arthropods like mosquitoes or ticks or direct contact with infected mammals [Tularemia is a zoonotic disease caused by the highly infectious, virulent, gram-negative bacterium involved . The geomisphere , and onlustralia , and Thaustralia . F. tulapathogen . Whereas mammals ,6.F. tularensis was the subject in diverse military biological weapons programs and is still included among the top six agents with high potential to be misused in bioterrorism [Due to its infectious nature, ease of dissemination and high case fatality rate especially in respiratory infection, errorism .F. tularensis is complex and has changed frequently. At present, the Francisellacae family contains four validly published species: F. tularensis, F. novicida, F. noatunensis and F. philomiragia. F. philomiragia is an opportunistic pathogen which has been rarely isolated from immuno-compromised individuals [F. noatunensis has been described to cause a granulomateous disease in fish [F. novicida was shown to be very closely related to F. tularensis, and most scientific authors consider it to be the fourth subspecies (subsp.) of F. tularensis (F. tularensis subsp. novicida) [Francisella species have been described [F. tularensis show close genetic and phenotypic relationship and have probably evolved from a common ancestor, they exhibit striking variation in virulence in humans and animals [F. tularensis subsp. tularensis (Type A), endemic in North America and F. tularensis subsp. holarctia (Type B) which is found in many countries of the holarctic region [tularensis can be further divided into two major clades, AI and AII, which seem to differ in virulence and to cause significant mortality differences in human infections [Francisella may comprise additional, hitherto unknown species [Francisella-like bacteria had been grown from immuno-compromised patients [Francisella species have been identified only by molecular means analyzing specimens from rodents, soil and water samples [Francisella-like bacteria have been found in diverse tick species and are believed to represent endosymbionts of arthropods [The taxonomic position of ividuals . F. noat in fish ,10. F. novicida) ,11. In tc region . Both suc region . Type A fections ,12. In a species -15. Whilpatients , some pu samples ,15. Morethropods .Francisella species or for a rapid and reliable discrimination of type A or type B tularemia. Cultivation of F. tularensis from clinical specimens requires at least two days; this is followed by detection of specific antigen, e.g. LPS and molecular typing. Some reports have identified unusual F. tularensis strains, isolated from patients or rodents, which lack cysteine requirement or production of regular F. tularensis LPS [F. tularensis may be difficult to recover in human and animal infection by using standard cultivation techniques, although direct immunofluorescence, immunohistochemical analysis or PCR allows detection of the organism within clinical samples [In clinical microbiology, the established cultivation and serological techniques are not sufficient for the diagnosis of all nsis LPS ,16,18. T samples -21.F. tularensis subspecies is important in the monitoring of enzootic tularemia during outbreaks of human tularemia or post-release scenarios [Rapid identification of cenarios ,22,23.Francisellae in clinical samples may prove helpful to both clinicians and public health authorities. Therefore, a 23S rRNA-based detection approach was developed, since this molecule has been used extensively to elucidate phylogenetic relationships of bacteria at intra- and intergeneric levels and it is also an excellent target for fluorescent in situ hybridization [Thus, rapid and reliable procedures for the direct detection and differentiation of dization -26.F. philomiragia and all four subspecies of F. tularensis were determined. Additional sequences for this target, which exists in three copies in the known Francisella genomes, were analyzed by extracting this information from the published whole genomes sequences currently available. These sequence data were used to develop additional primer sets and fluorescently labeled oligonucleotide probes suitable for species- and subspecies-specific fluorescent in situ hybridization (FISH) of pathogenic Francisella species in culture as well as clinical specimens.Near full-length 23S rRNA gene sequences for Francisella strains were grown aerobically on heart cysteine agar (HCA) at 37°C and 5% CO2. All other strains were cultured on Columbia blood agar or in Luria-Bertani (LB) broth . Bacterial cells were harvested while in exponential phase, suspended in phosphate buffered saline (PBS), centrifuged, washed in PBS, resuspended in TE buffer , and adjusted to an optical density of 1.0 at 600 nm. Bacterial suspensions were prepared for PCR analysis using the QIAGEN  tissue kit as recommended by the manufacturer. For in situ hybridization, harvested cells were processed and fixed with paraformaldehyde (PFA) as previously described [All bacterial strains used in this study are listed in Table escribed .F. tularensis subspecies as well as one strain of the species F. philomiragia were performed as described by Lane [Amplification and sequencing with universal primers of one strain of each  by Lane . Full leFrancisella genomes were used for the design of new PCR primers and hybridization probes. Primer and probe designations, sequences, positions, and references are listed in Additional file Francisella isolates comprising strains of each F. tularensis subspecies. For each PCR, 2 μL of DNA extracts were used. PCR was performed in a total volume of 50 μL containing 20 μL 5-Prime-MasterMix 2.5×  and 0.2 μM of each the forward and reverse primer. The PCR were performed with a GenAmp PCR System 9700 thermocyler . Cycling conditions were: Initial denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 40 seconds, annealing at 56°C for 30 seconds and amplification at 72°C for 90 seconds and a single final extension at 72°C for 5 minutes. The PCR products were purified with the QIAquick PCR Purification Kit™ according to the manufacturer's manual .The alignment of all five complete 23S rRNA gene sequences and additional six sequences from publicly available whole ® Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems . The total volume of the sequencing reaction-mix was 10 μL containing 4 μL of the ready mix (BigDye® Terminator v3.1 Cycle RR-100) from the kit, 3 μL of the purfied PCR product and 0.2 μM of the respective sequencing primer. Identical primers were used in both the amplification and sequencing PCR. All sequencing reactions were performed with the same thermocycler as described above. Cycler conditions were: An initial denaturation step at 96°C for 1 minute, followed by 25 cycles of denaturation at 96°C for 10 seconds, annealing at 50°C for 5 seconds, and extension at 60°C for 4 minutes. The sequencing products were purified with Centri-Sep spin columns  and subsequently analyzed on an 3130 Genetic Analyzer  in accordance with the instructions of the manufacturer.The purified PCR products were sequenced with an BigDyehttp://www.arb-home.de and probeBase http://www.microbial-ecology.net/probebase, synthesized and tagged with 6-FAM or Cy3 fluorescence dyes .Genus-, species- and subspecies-specific oligonucleotide probes for fluorescent insitu hybridization were developed using the software package ARB Francisella species or subspecies, two probe combinations were hybridized simultaneously to the reference cells and to the clinical samples in most cases. While testing the specificity and sensitivity of the newly developed probes, each preparation of reference cells from all different bacterial strains were additionally probed with a generic eubacterial probe (EUB338) and a non-sense nucleotide probe (NonEUB338) to confirm accessibility of the target rRNA as well as to exclude unspecific labelling of bacterial cells or tissue due to preparation artefacts [Francisella species and to discriminate between F. philomiragia and F. tularensis. The combination of probes Bwtume168II and Bwmed1397 was applied in order to identify and discriminate F. tularensis subsp. tularensis (type A) and F. tularensis subsp. mediasiatica. Isolates of the subspecies F. tularensis holarctica and F. tularensis subsp. novicida were identified using probes Bwhol1151 or Bwnov168, respectively.In situ hybridization on glass slides was performed as described previously . For thertefacts . Probes The addition of 30, 35 or 50% formamide to the hybridization buffer resulted in specific hybridization of the oligonucleotides to their respective target organisms. To reduce the amount of toxic waste, formamide was not used in the washing steps following hybridization. As a substitute, the NaCl concentration was decreased in the washing buffer according to the formula of Lathe [F. tularensis subspecies. Single cultures were started with inoculums of 10 to 1000 colony forming units (cfu) in 5 ml whole human blood. Additionally, cells from two different subspecies were mixed at ratios of 1:1, 1:10, 1:100, 1:1000 and then cultured under aerobic conditions until the BACTEC instrument reported bacterial growth. Aliquots of 10 to 40 μl were dropped onto glass slides, air-dried and then fixed with PFA as described above.Aerobic BACTEC blood culture bottles  were spiked with live bacterial cells from different F. tularensis subsp. holarctica and F. tularensis subsp. novicida at a multiplicity of infection (MOI) of 100, incubated for 120 minutes and then fixed with paraformaldehyde [Two different cell lines, the human monocyte/macrophage lineage U937 and the mouse macrophage cell line J 774 were infected with aldehyde . Paraffi23S rRNA gene sequences of F. tularensis subsp. mediasiatica Francisella Strain Collection (FSC) 147, F. tularensis subsp. tularensis Schu S4, F. philomiragia ATCC 25017, F. tularensis subsp. holarctica ATCC 29684, and F. tularensis subsp. novicida ATCC 15482, have been deposited under accession numbers GU073995 to GU073998 and GU073986, respectively.The nearly complete 23S rRNA gene sequences of 24 additional Francisella strains have been deposited under accession numbers GU073970 to GU073985, and GU073987 to GU073994.The partial F. tularensis subsp. tularensis Schu S4, F. tularensis subsp. holarctica ATCC 29684, F. tularensis subsp. mediasiatica FSC 147, F. tularensis subsp. novicida ATCC 15482, and F. philomiragia ATCC 25017. The 23S rRNA gene sequences of the F. tularensis subspecies exhibited very high levels of homology (99.4 to 99.9% identity). Between F. tularensis subsp. tularensis FSC 237 (Schu S4) and F. tularensis subsp. holarctica  11 different single nucleotide substitutions were found. Differences between F. tularensis subsp. novicida (ATCC 15482) and the three other subspecies ranged from 10 to 19 single nucleotide substitutions.The PCR primers 630V, 985R, 1029V and 502RN directed the synthesis of two overlapping 23S rRNA gene fragments, which covered the complete 23S rRNA gene , labeled with a different fluorochrome allowed the identification of all investigated Francisella strains on the species level by a single analysis  is complementary to all F. tularensis holarctica sequences and gave strong signals when hybridized to its respective target strains  or only to the 23S rRNA of F. tularensis subsp.mediasiatica (Bwmed1379)  Fig. . The firse Table .F. tularensis subsp. holarctica and F. tularensis subsp. novicida could then be identified by hybridization with their specific probes  was used to grow bacterial cells from each representative strain initially used for 23S rRNA gene sequencing. The culture bottles were spiked with 5 ml of human blood and the bacteria grown on HCA medium. Depending on the subspecies and the initial inoculum size, growth in aerobic blood culture bottles occurred between two to eleven days of incubation. Bacterial cells from each subspecies were strongly labeled with their corresponding probes as well as the EUB338 probe used for positive control  both populations could be easily separated by whole cell hybridization with distinctly labeled probes Fig. . By thisF. tularensis, is regarded as a potential biological warfare or bioterrorism agent of the highest category. For these reasons clinical and public health laboratories are urged to provide rapid and reliable diagnostic tools for the sensitive detection and identification of F. tularensis and for the appropriate differentiation of its subspecies or clades which often display significantly divergent pathogenic potential [Tularemia is a rare but dangerous zoonosis, which is endemic in almost all countries of the Northern Hemisphere. In some areas like Central and Southern Europe as well as Turkey, tularemia is an emerging or re-emerging disease representing a significant threat for public health -35. Its otential ,22.F. tularensis is difficult due to its fastidious nature and its susceptibility to overgrowth by concomitant flora. Additionally, growth may be delayed (up to 12 days) and cultivation of F. tularensis poses a significant threat of laboratory infections. Only recently, conventional and real-time PCR protocols for the detection and identification of F. tularensis have been published, but still none of these techniques is sufficiently evaluated to be routinely used in clinical laboratories [In contrast to most other bacterial pathogens, cultivation of ratories .Francisella species. In- silico analysis of partial and complete 16S rRNA genes available in publicly accessible databases like GenBank confirmed the results of a previous study by showing that 16S rRNA sequences from F. tularensis subspecies are almost identical, and therefore, are only of limited value for the detection and discrimination of F. tularensis on the species or subspecies level [Yersinia (Y.) pseudotuberculosis and Y. pestis or Burkholderia (B.) pseudomallei and B. mallei [In this study we evaluated the potential of rRNA gene targeted PCR and sequencing as well as fluorescent in situ hybridization for the detection and differentiation of es level . In this. mallei ,37,38F. tularensis subspecies as well as F. philomiragia revealed several discriminative SNPs. The sequence data obtained from rRNA gene sequences, known to be highly conserved in bacterial phylogeny, could be successfully used for the construction of hybridization probes, allowing a rapid genotype-based detection of Francisella species on different taxonomic levels.In contrast to those studies, comparison of full-length 23S rRNA genes of all F. tularensis subsp. holarctica (type B) could be identified. For the discrimination of F. tularensis subsp. tularensis (type A) and subsp. mediasiatica, an identification approach was developed by employing two different probes. Six type A strains, 31 type B strains as well as three F. tularensis subsp. mediasiatica strains were correctly identified by this approach, whereas no false-positive signal was observed with 71 other variably related bacterial species.A unique 23S rRNA target region suitable for the detection of F. philomiragia and F. tularensis, which were tested with all mentioned F. tularensis strains as well as four F. philomiragia strains. We also developed an in situ hybridization protocol for F. tularensis subsp. novicida, which allowed the detection of all four available strains of this subspecies. However, due to the possible genetic variability within this subspecies [novicida-like' strains [23S rRNA gene sequences of the aforementioned fish pathogenic members of the genus Francisella became publicly available [F. noatunensis will be probably detected by probe Bwall1448. The available data also indicate, that at it might be possible to discriminate between F. noatunensis comp. nov. and F. noatunensis subsp. orientalis if probe Bwphi1448 would be combined with probe Bwall1448. It is mandatory to experimentally verify these sequence-based predictions.Similar results were gained employing species-specific probes for bspecies  it remai strains ,4,13-16.vailable ,10. An iF. tularensis, discrimination of bacterial strains to the subspecies level by means of conventional PCR was almost impossible until 2003 [Caused by the genetic homogeneity and the clonal population structure of til 2003 . Today, til 2003 ,42. HoweF. tularensis LPS antibodies is routinely applied [Fluorescent oligonucleotide probing of whole cells is fast (less than two hours), reliable and could be analyzed by regular fluorescence microscopy, which is available in virtually all clinical or public health laboratories. In tularemia, immunofluorescence staining of clinical samples with anti- applied , but ant applied .Pseudomonas aeruginosa, B. cepacia) in typical patient specimens such as sputum or blood culture [For two additional reasons, fluorescent in situ hybridization is a suitable alternative to biochemical identification or PCR. First, it can be applied to thoroughly inactivated clinical or culture samples thereby reducing the threat of laboratory infection. Second, it works without expensive and technical sophisticated devices, rendering FISH a cost-effective procedure. The potential for routine application of this method is supported by the availability of commercial test kits for clinically relevant species . Table S2: Subspecies specific single nucleotide polymorphisms (SNPs) in the sequence of the 23S rRNA gene based on sequences of 29 Francisella strains.Click here for file