research article xenic cultivation and genotyping of

Research ArticleXenic Cultivation and Genotyping of Pathogenic Free-LivingAmoeba from Public Water Supply Sources in Uganda

Celsus Sente1 Joseph Erume2 Irene Naigaga1 Benigna Gabriela Namara3

Julius Mulindwa24 Sylvester Ochwo2 Phillip Kimuda Magambo2 Charles Drago Kato2

Andrew Tamale1 and Michael Ocaido1

1Department of Wildlife and Aquatic Animal Resources (WAAR) School of Veterinary Medicine and Animal Resources (SVAR)College of Veterinary Medicine Animal Resources and Biosecurity (COVAB) Makerere University PO Box 7062 Kampala Uganda2Department of Biomolecular Resources and Biolab Sciences School of Biosecurity Biotechnical and Laboratory Sciences (SBLS)College of Veterinary Medicine Animal Resources and Biosecurity (COVAB) Makerere University PO Box 7062 Kampala Uganda3Research Unit on AIDS Medical Research Council (MRC)Uganda Virus Research Institute (UVRI) PO Box 49 Entebbe Uganda4Department of Biochemistry and Sports Science (BSS) College of Natural Sciences (CONAS) Kampala Uganda

Correspondence should be addressed to Celsus Sente csente37gmailcom

Received 17 May 2016 Accepted 18 July 2016

Academic Editor Xinhua Shu

Copyright copy 2016 Celsus Sente et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Studies on waterborne parasites from natural environment and domestic water sources in Uganda are very scarce and unpublishedWater dwelling free-living amoebae (FLA) of the genus Acanthamoeba Hartmannella and Naegleria are often responsible forcausing morbidities and mortalities in individuals with recent contact with contaminated water but their presence in Ugandarsquospublic water supply sources is not known We cultivated and genotyped FLA from natural and domestic water from QueenElizabeth Protected Area (QEPA) and Kampala (KLA) The cultivated parasites were observed microscopically and recordedThe overall prevalence of FLA in QEPA (Acanthamoeba spp 35 Hartmannella spp 189 Naegleria spp 135) and KLA(Acanthamoeba spp 283Naegleria spp 166Hartmannella spp 231)were not significantly differentThe highest prevalenceacross water sources in QEPA and KLA was observed for Acanthamoeba spp followed by Hartmannella spp and Naegleria sppOverall FLA mean (plusmnSE) and mean (plusmnSE) across water sources were highest for Acanthamoeba spp compared to other FLA butwere not statistically significant (p gt 005) Analysis of the FLA sequences produced 1 Cercomonas 1 Nuclearia 1 Bodomorpha 2Hartmannella 5 Echinamoeba and 7 Acanthamoeba partial sequences indicating a muliplicity of water contaminants that need tobe controlled by proper water treatment

1 Introduction

Free-living amoeba (FLA) of the genera Naegleria Acan-thamoeba and Hartmannella have been associated withwater-related illnesses especially in developed countriesfound in America Europe and the Middle East [1ndash4] Inmany parts of Uganda there is dependence on environmentalwater for washing recreation drinking cooking and otherdomestic purposes [5] However in most cases this water isused without treatment an act which predisposes Ugandansto FLA infection [5] Since some of these amoebae are knownto be fatal it is imperative that their occurrence is monitored

in order to assess the human and probably animal risksinvolved

Studies worldwide have documented the common Nae-gleria Acanthamoeba and Hartmannella species associatedwith infectious diseases resulting from contaminated watersources utilised by communities Considering the large num-bers ofNaegleria species it is onlyN fowleriN australiensisand N italica that are considered to be pathogenic [6ndash8] while other species such as N gruberi N jadini Nlovaniensis N indonesiensis N robinsoni N fultoni and Npagei have not yet been reported as pathogenic [8] Infor-mation also denotes Acanthamoeba polyphaga A hatchetti

Hindawi Publishing CorporationNew Journal of ScienceVolume 2016 Article ID 6358315 9 pageshttpdxdoiorg10115520166358315

2 New Journal of Science

A castellanii A culbertsoni A rhysodes A lugdunensisA quina A griffini Balamuthia mandrillaris Hartmannellavermiformis and Vahlkampfia avara as pathogenic speciesmostly associated with keratitis in humans [9 10] Acan-thamoeba spp particularly renders amplification of Vibriocholerae Legionella pneumophila Bacillus anthracis andMycoplasma tuberculosis inside their cells [10ndash12] a propertyof paramount importance to both humans and wildlife inand around QEPA Hartmannella spp that have in manyinstances been isolated in mixed human amoebic keratitisinfections are recently considered to be very pathogenic FLA[13] This work therefore reports the different pathogenicFLA associated with tap and environmental surface water inQEPA and Kampala an indicator towards water toxicity forthe communities In the past 10 years outbreaks of choleratyphoid and other waterborne diseases have been docu-mented in the study areas [14ndash17] and these could be linkedto distribution by contaminated water sources as well

The FLA of the genera Naegleria Acanthamoeba andHartmannella predispose humans who collect water fromvarious sources to diseases [1 10 18] These pathogenic andopportunistic FLA are aerobic eukaryotic protists that occurworldwide and can potentially cause infections in humansand other animals [10 19 20] The bacterial parasites Vibriocholerae Legionella pneumophila Mycoplasma tuberculosisand Bacillus anthracis are easily amplified within certainAcanthamoeba spp Hartmannella spp Naegleria spp andother FLA consequently increasing their potential to causecholera legionellosis tuberculosis and anthrax respectively[10ndash12 19 20]

The risks associated with FLA infection inUganda are notknown the reason why this study is being carried out Theassociations are expected to be high because significant por-tions of these communities are using natural environmentalwater and domestic tap water sources without any definitemeasures for protecting water systems in QEPA and KLAfrom these organisms [5] FLA are not often mentioned aspossible infectious agents yet they have severe pathogeniceffects [25 26] Disease effects associated with pathogenicFLA in humans and animals often go undetected becausethere is scarce information about their distribution InUganda the gap being addressed is that of studyingpathogenic FLA in environmental water and domestic tapwater systems This information will help explain the risksassociatedwithwater-related illnesses among rural and urbandwelling Ugandans

2 Methods

21 Study Areas The study was conducted in a rural areaQueen Elizabeth Protected Area (QEPA) in western Ugandaand an urban area Kampala (KLA) City Uganda TheQEPA is located at 00 12S 30 00E (latitude 02000 lon-gitude 300000) and is 1978 sq km in size The pro-tected area is known for its wildlife including Cape buf-faloes (Syncerus caffer caffer) Hippopotami (Hippopotamusamphibius) Nile Crocodiles (Crocodylus niloticus) elephants

(Loxodonta africana) leopards (Panthera pardus) lions (Pan-thera leo) chimpanzees (Pan troglodytes) and Uganda kobs(Kobus kob thomasi) It is a home to 95 species of mammalsand more than 500 species of birds The protected area isalso famous for its volcanic features including volcanic conesand deep craters QEPA is a UNESCO ldquoMan and BiosphereReserverdquo with 11 village enclaves all with a fast growing pop-ulation of humanswhosemain economic activities are fishingand livestock production The diversity of animals in theprotected area coupled with adjacent human communitiesmakes it a vital hub for sharing of infections at water pointsources

Kampala is the capital city of Uganda lying at latitude0∘1810158405810158401015840N longitude 32∘3410158405510158401015840E with 7297mi2 (189 km2)and divided into five boroughs each consisting of a con-centration of slums with a rapidly growing populationCurrently estimated at 1659600 [27] Kampalarsquos population isincreasing steadily yet service provision is not improvingWater resources used in the city vary from piped waterprotected springs unprotected springs and natural surfacewater (lakes rivers streams swamps roadside gutters andpasture puddles) which are often relatively unsafe for humanconsumption

22 Ethical Consideration This study does not require anethical statement

23 Study Design The study involved one year of field workand one year of confirmatory laboratory phase Laboratoryphase involved analysing water samples for FLA The watersources considered were those from natural environmentaland domestic tap water sources The sampling sites werepurposively selected based on their benefit convenience andimportance to public health

In QEPA selection of the study area was based on certainlandmarks that included the following (1) along the Kyam-buraRiver (2)KazingaChannel banks (3)KazingaMidchan-nel (4) fish landing sites (FLS) and (5) community pipedtap water In Kampala the location of sampling sites wasbased on previous waterborne disease outbreak occurrencesWe selected areas in central Kampala (Banda KisenyiKatanga Kasubi Kazo Bwaise Lubigi and Makerere) thatwere reported to have undergone disease outbreak in the last 5years and collected samples from piped tap water and naturalenvironmental surface water (swamp and stream)

24 Laboratory Methods

241 Xenic Cultivation of FLA from Water Samples Weused nonnutritive medium (Page Amoeba Saline solution of25mM NaCl 1mM KH

2PO4 05mM Na

2HPO4 40mM

CaCl2 and 20mM MgSO

4) seeded with 01mL of a heat

inactivated 48-hour culture of Escherichia coli BL2 [5 28]Fresh water samples in the 50mL tubes were centrifuged at1000timesg for 15 minutes and supernatant was poured off toexpose the pellets Using sterile Pasteur pipettes the pelletswere removed from all the tubes and each was carefullyspreadplated on preseeded NNA-EI agar plates This was

New Journal of Science 3

followed by incubating the plates at 32∘C overnight afterwhich each plate was sealed with a plastic film and incubatedupside down at 32∘C up to 7 days Three days later theplates were monitored for detection of amoebae trophozoitesuntil day 7 using an inverted microscope (Motic AE2000Binocular TED PELLA Inc USA)

242 DNA Extraction All DNA was extracted from culturepositive plates by chemical lysis and purification [29] Fivehundred microliters of STE buffer (01M NaCl 1mM EDTA10mM trischloride PH 8 and 1 SDS) and 10 120583L proteinaseK (10mgmL) were added to each sample in an Eppendorftube All samples were put in a water bath and incubated at56∘C for one hour and then left to cool down before phenolextraction Phenol chloroform (521120583L) was added to eachsample vortexed and centrifuged at maximum speed(13200 rpm) for 5 minutesThe aqueous layer was transferredto a new Eppendorf tube and the step redone to make 2phenol-chloroform extractions The aqueous layer was sub-jected to another chloroform extraction centrifuged andtransferred to a new Eppendorf tube after which 1000120583L ofabsolute alcohol (96ndash100) was added to each sample Thesamples were put in a freezer at minus80∘C overnight for pre-cipitation The following day all samples were centrifuged at13200 rpm for 30 minutes and alcohol was poured offThe pellet was washed with 1000 120583L of 70 alcohol andcentrifuged at 13200 rpm for 15 minutes Alcohol was pouredoff to expose the pellet which was air-dried and dissolved in50120583L of TE buffer

243 DNA Amplification Amplification of 18S ribosomalDNA (18S rDNA) from Acanthamoeba and other FLA wasdone by primer pairs JDP1JDP2 and CRN51137 [12 30 31]Forward primer JDP1 (51015840GGCCCAGATCGTTTACCG-TGAA-31015840) and reverse primer JDP2 (51015840TCTCACAAG-CTGCTAGGGAGTCA-31015840) were genus-specific for Acan-thamoeba spp Forward primer CRN5 (51015840CTGGTTGAT-CCTGCCAGTAG-31015840) and reverse primer 1137 (51015840GTGCCC-TTCCGTCAAT-31015840) obtained amplimers from any eukaryoteaiding amplification of the 18S ribosomal DNA gene fromdifferent FLA

The reactions were carried out with a DreamTaq PCR kit(Thermo Scientific DreamTaq USA) A 25 120583L reaction vol-ume containing 125 120583L DreamTaq Green PCR Master Mix(2x) 05 120583M forward primer 05120583M reverse primer 9120583Lnuclease-free water and 25 120583LDNA template (50 pg concen-tration) was used The following conditions were consideredfor the PCR initial denaturation at 94∘C for 3 minutesfollowed by 35 cycles with denaturation at 94∘C for 30seconds followed by annealing at 55∘C for 30 seconds thenextension at 72∘C for 30 seconds and a final extension at72∘C for 5 minutes Five microliters of each PCR reactionwas tested for successful amplification using agarose gel(25WV) stained with ethidium bromide run against1 kbp DNA ladder Once enough electrophoretic separationwas reached the agarose gel was observed under a UV geldocumentation system thereafter capturing the gel images

Dry season Rainy season Dry season Rainy season

QEPA KLA

Acanthamoeba sppHartmannella spp

Naegleria sppOther FLA

(n = 204) (n = 174)(n = 116)(n = 204)

010203040506070

Ove

rall

prev

alen

ce

Figure 1 Overall seasonal prevalence of FLA

25 Nucleic Acid Sequencing and Analysis Positive gel sam-ples were extracted and the DNA purified with QIAquick GelExtraction Kit (Qiagen Inc Netherlands) Partial 18S rDNAsegment of the amoeba isolates was exposed to cycle sequenc-ing with JDP1JDP2 and CRN51137 as sequencing primers[31] Base trimming of the sequence files to obtain goodquality was done by ldquoSeqBuilderrdquo software (Dnastar USA)and a search for homologues in the NCBI database was doneusing ldquoblastnrdquo tool The resultant homologues with querycoverage gt 70 identity gt 70 and low E values (lt0)were considered Phylogenetic analysis was done and adendrogram was constructed [32]

26 Statistical Analysis Data was entered into Excel fromwhich it was extracted and analysed using SPSS (IBM USA)Variables were summarised by the use of mean and standarderror of themean (SEM) Application of univariate analysis tocompare prevalence across sampling sites was executed usingcross-tabulation with a 1205942 test All variables with a 119901 valueof le 005 were considered significant We employed Pearsoncorrelation coefficient (r) to carry out linear correlation anal-ysis between naturaltap water variables and water parasitepresence

3 Results

31 Prevalence of Parasites

311 Overall Seasonal Prevalence The water samples werecollected during cold rainy (November March and July) andcool dry (January May and September) seasons OverallFLA parasite prevalence in both study sites was higher duringthe rainy season except for Naegleria spp that was higher inthe dry season in QEPA (Figure 1)

312 Overall Prevalence andMean Theprevalence andmean(SEM) of the parasites from different sources are shownin Table 1 Both natural environmental and domestic tapwater sources were contaminated with FLA The overallprevalence of Acanthamoeba spp and other FLA in QEPAand Kampala was as follows Acanthamoeba spp (QEPA


Table 1 The overall prevalence of the FLA

Parasite QEPA KLAFreq (119899 = 408) Prev () Mean plusmn SE Freq (119899 = 290) Prev () Mean plusmn SE

Acanthamoeba spp 143 35 19 plusmn 02 82 283 28 plusmn 05Naegleria spp 55 135 04 plusmn 01 48 166 14 plusmn 03Hartmannella spp 77 189 12 plusmn 01 67 231 14 plusmn 03Freq frequency Prev prevalence

Table 2 Prevalence and mean across water sources

Parasite QEPA KLATap water (119899 = 84) Environmental water (119899 = 324) Tap water (119899 = 170) Environmental water (119899 = 65)

Acanthamoeba spp(+) () 36 (43) 107 (33) 48 (282) 15 (231)Mean plusmn SEM 226 plusmn 04 892 plusmn 16 52 plusmn 12 24 plusmn 13

Hartmannella spp(+) () 19 (226) 58 (179) 38 (224) 10 (154)Mean plusmn SEM 120 plusmn 015 493 plusmn 092 44 plusmn 10 09 plusmn 016

Naegleria spp(+) () 12 (143) 43 (133) 25 (147) 9 (138)Mean plusmn SEM 05 plusmn 015 158 plusmn 06 35 plusmn 11 05 plusmn 03

35 KLA 283) Hartmannella spp (QEPA 189 KLA231) and Naegleria spp (QEPA 135 KLA 166) Themean (plusmnSEM) was highest for Acanthamoeba spp followedby Hartmannella spp and lastly Naegleria spp

313 Prevalence of Parasites across Sampling Sites Prevalenceof parasites across water source (natural and tap water)considered is presented in Table 2 Acanthamoeba spp werethe most prevalent parasite across all sources Naturalenvironmental water had significantly higher mean valuescompared to tap water in both study sites

314 Molecular Identification and Phylogenetic Analysis Fol-lowing FLA sequencing the products were blasted andcompared with the GenBank results from NCBI (Figure 2Table 3) Comparisons between the FLA isolated in Ugandaand those from the NCBI database were made and dif-ferences in divergence noted (Figure 2 Table 4) wereassessedThe species identifiedwereAcanthamoeba spp (var-ious T-genotypes) Acanthamoeba polyphaga Hartmannellavermiformis Nuclearia pattersoni Echinamoeba exundansBodomorpha minima and Cercomonas agilis The Acan-thamoeba sequences got belonged to the group of sequencetypes T1 T2 T4 and T11 (Table 3)

The confirmed Acanthamoeba genotype T1 was isolatedfrom tap water in Bwaise whereas T2 and T11 were isolatedfrom the Kazinga Channel water in QEPA Genotype T4which is usually the commonest Acanthamoeba T-genotypewas isolated from tap water in Katunguru Trading CentreKasaka landing site Albertine Restaurant in QEPA and

Kisenyi slum in Kampala The Hartmannella vermiformisconfirmed was isolated from fish landing sites in QEPA Thenonpathogenic FLA (Table 3) were isolated from a variety oftap water samples in Albertine Restaurant Thembo Restau-rant andKatunguruTradingCenter aswell as natural surfacewater from fish landing sites and the Kyambura River inQEPA In Kampala natural water samples that were positivefor FLA were from Lubigi swamps

All the parasites identified in this study were matchedwith the reported diseases they cause in humans (Table 3)

4 Discussion

We investigated the presence of FLA in natural and domestic(tap) water in QEPA and KLA The prevalence and mean(plusmnSEM) of Acanthamoeba spp in all cases were higher thanNaegleria and Hartmannella spp All FLA were more preva-lent in the rainy season exceptNaegleria spp that were higherin the dry season Most waterborne parasites increase innumber during the rainy season due to contamination of thewater sources with sewage soil and other organic mattersfrom water run-off [5 33] Tap water had a higher prevalenceof FLA than the natural water source There is not muchdata in Uganda to compare with the present findings butstudies from other countries [34 35] also documented ahigher frequency of Acanthamoeba compared to other FLAin environmental and tap water samples in alkaline water atthe same temperaturesThe first ever study of FLA in Ugandadocumented higher prevalence and mean of Acanthamoebathan other FLA in both environmental and tap water [5]


1

02

072

008

009

004

006

012

014

089

0861

0

10

078

087

077

091

077

091

018

058

063

054

088

033

085

KU894812

AF2938951_Echinamoeba_exundansKU894811

KU894808

KU894813

KU894806

AJ4892611_Echinamoeba_thermarumKU894805

KU894814

JQ2716891_Hartmannella_vermiformisKT1856251_Vermamoeba_vermiformisKU894803

KU894799

KU894800

KU894801

KU894802

KU894804

KM1894191_Acanthamoeba_spKU894807

AY3646351_Nuclearia_pattersoniAF4846871_Nuclearia_simplexKU894810

AY7488061_Cercomonas_agilisEU7091401_Cercozoa_spEU6471741|_CercomonadidaKU894809

AF4112761_Bodomorpha_minimaKU884884

Figure 2 Phylogenetic tree based on neighbor-joining showing the divergence of FLA Comparison with closely related species from theGenBank database sequences with their accession numbers (GenBank NCBI)

Acanthamoeba spp are more commonly encountered prob-ably because they are more involved in a predator-preyrelationshipwithmicrobial coloniesThehigh numbers couldbe explained by the presence of organic matter from rottingleaves animal and human faeces which are from the run-off from the land that often concentrate at the banks of thewater bodies This is known to exacerbate microbial biofilmformation and as a result facilitate the proliferation of FLAIt is believed that there are more FLA when there is anaccumulation of more organic matter in soil and water [3637] Previous studies report thatmicroorganisms settle on theinner surfaces of water pipes later becoming a source ofsecondary microbial contamination [38]

The primer pair JDP1JDP2 that was used is more spe-cific in the amplification of Acanthamoeba DNA [30 39]whereas CRN51137 amplifies any eukaryoteDNA [30]Acan-thamoeba sequence types can be grouped T1 to T20 [40]Blasting of sequences and comparison with those fromNCBIdatabase produced 7 Acanthamoeba 5 Echinamoeba 2 Hart-mannella 1 Bodomorpha 1 Nuclearia and 1 Cercomonas par-tial sequences Acanthamoeba genotypes T1 T2 and T4 were

mainly isolated from tap water samples whereas 11 were iso-lated from environmental water samplesThis is in agreementwith previous studies that indicate that T2 Acanthamoebagenotype is mainly found in the environment and is phyloge-netically related to T6 and they have also been both isolatedfrom clinical AK cases in humans [40] Acanthamoebagenotype T11 is closely related to T4 and has been found toalso cause AK [33]Acanthamoeba of T4 genotype is reportedas the most commonly encountered T-genotype group inboth environmental water and clinical samples and also themost diverse [33] Genotype T1 is notorious for granulo-matous amoebic encephalitis [11] whereas others may causekeratitis cutaneous infections and sinusitis in humans [1841] Hartmannella vermiformis originally thought to be non-pathogenic [42] has over the past decade been repeatedlyreported in a number of mixed human AK infections [4 1324] Although other FLA isolated in this study are considerednonpathogenic it is possible that they too could becomevirulent anytime given a conducive environment

Infective trophozoites of FLA in the environment aremaintained and spread by water during rainy seasons when


Table 3 Isolated free-living amoeba accession numbers water source and associated diseases in humans

Accession Source FLA Diseases Characteristics

KU884884 Tap KLA Bwaise Acanthamoeba spp (T1) Encephalitis [11 21]Mental status changeshemiparesis meningismus andataxia [22]

KU894799 Tap QEPA Katunguru Acanthamoeba spp (T2) Keratitis [11 21] Blinding infection of the cornea[22 23]


KU894801 Tap QEPA Albertine Acanthamoeba polyphaga(T4) Keratitis [11 21] Blinding infection of the cornea

[22 23]

KU894802 Tap QEPA Kasaka Acanthamoeba spp (T4) Keratitis [11 21] Blinding infection of the cornea[22 23]

KU894803 Tap KLA Kisenyi Acanthamoeba spp (T4) Keratitis [11 21] Blinding infection of the cornea[22 23]

KU894804 KCB QEPA Acanthamoeba spp (T11) Keratitis andencephalitis [22]

Disseminated disease blindnessand CNS function impairment[22]

KU894805 FLS QEPA Hartmannella vermiformis Keratitis [4 13 24] Blinding infection of the cornea[22]

KU894806 TAP QEPA Albertine Echinamoeba exundans Unknown mdashKU894807 TAP QEPAThembo Nuclearia pattersoni Unknown mdashKU894808 TAP QEPA Katunguru Echinamoeba exundans Unknown mdashKU894809 FLS QEPA Bodomorpha minima Unknown mdashKU894810 Lubigi KLA Cercomonas agilis Unknown mdashKU894811 R Kyambura QEPA Echinamoeba exundans Unknown mdashKU894812 R Kyambura QEPA Echinamoeba exundans Unknown mdashKU894813 TAP QEPA Albertine Echinamoeba exundans Unknown mdash

KU894814 TAP QEPAThembo Hartmannella vermiformis Keratitis [4 13 24] Blinding infection of the cornea[22]

FLS fish landing sites KCB Kazinga Channel banks KLA Kampala QEPA Queen Elizabeth Protected Area R Kyambura the Kyambura River

there is a run-off of water containing human and animal fae-cal matter from land into the water bodies which eventuallyend up at the points where communities fetch and utilise thewater Most FLA prevalence and mean intensities are higherin the rainy season than dry season [43] However parasiteincidences can be high throughout the rainy and dry seasonsoften indicating poor disposal of human and animal excretaand continuous patterns of infection [43] In a natural waterenvironment pathogens have been isolated widely frommany water sources used by rural dwelling households [3344 45] Often inadequately treated domestic water (drinkingbathing cooking and recreational water) has an abundanceof such pathogens Water bodies are usually contaminatedby high concentrations organisms from agricultural run-offurban wastewater effluents [46] and for the case of QEPAandKLAhuman and animal faecal contaminationTheQEPAand KLA local communities have few poorly built latrinesmost of which are already filled up compelling many to digsmall holes in the ground and defecate outside on openland When there is a heavy downpour of rain humanand animal the faecal material is washed off into the publicwater supply system Upon using this water exposure to avariety of protozoan parasites is highly likely The risk of

human infection is much higher in children and immuno-compromised individuals such as those who haveHIVAIDSdiabetes and cancer and those who have recently undergoneorgan transport [3]

5 Conclusion

The findings from the present study indicate that there isreasonably high contamination of both natural and domesticwater systems with Acanthamoeba spp Hartmannella sppNaegleria spp and other FLAThis is evidence that the waterbeing used is of poor quality and predisposes communities toinfectious agents With the fact that there is proof thatsome are pathogenic and can be vectors of many emerg-ingreemerging infectious agents it is imperative to preventthem from contaminating domestic water sources

Abbreviations∘C CelsiusAK Amoebic keratitisCNS Central nervous systemEnviron Environment


Table4Estim

ationof

evolutionary

divergence

betweensequ

ences

Percentidentity

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

Divergence

1532

536

534

528

538

538

514

521

565

345

360

315

522

256

533

254

537

252

251

248

243

250

252

305

299

305

309

1KU

884884

22391

973

956

960

944

944

533

554

549

329

346

307

554

264

525

239

915

238

235

256

242

246

246

293

289

299

310

2KU

894799

32203

19957

950

940

945

523

544

550

328

348

306

545

266

525

238

917

236

234

255

240

243

247

291

289

298

311

3KU

894800

42051

30

37

941

934

956

528

552

550

323

346

306

550

264

525

242

898

238

235

255

240

248

252

294

290

301

311

4KU

894801

52187

40

42

33

940

944

529

550

544

333

342

303

550

266

526

236

897

236

234

256

239

244

248

291

289

299

311

5KU

894802

62120

48

60

53

40

940

521

540

558

342

356

318

540

278

538

247

887

243

240

264

250

250

255

298

294

305

317

6KU

894803

72262

33

45

1743

57

509

530

554

332

353

315

530

275

536

251

890

246

243

263

248

254

258

299

298

307

326

7KU

894804

81310

1459

1409

1424

1447

1441

1388

795

632

357

370

329

807

277

546

283

562

277

277

256

256

256

264

302

299

305

310

8KU

894805

91271

1043

1030

1014

1032

1048

1006

484

659

455

420

362

960

430

666

322

583

323

322

416

381

315

322

356

353

364

352

9KU

894806

101335

1819

1777

1637

1793

1671

1584

500

389

474

537

491

670

415

666

389

581

381

383

395

395

479

486

471

477

482

485

10KU

894807

111410

1653

1636

1653

1490

1472

1564

640

264

483

652

596

478

677

651

553

313

545

545

654

585

549

556

588

591

592

589

11KU894808

121368

1691

1709

1633

1677

1562

1534

531

342

316

424

836

432

695

583

685

333

689

689

689

675

733

742

928

898

863

846

12KU

894809

131216

1391

1404

1392

1384

1332

1314

472

338

304

422

79374

701

534

697

291

683

683

681

664

744

752

847

860

864

958

13KU

894810

141279

1029

1026

1014

1017

1034

987

456

69

329

211

294

296

452

687

328

584

325

323

436

368

323

332

365

362

369

360

14KU

894811

151330

1238

1257

1247

1192

1156

1180

463

66

362

248

241

272

00

648

742

247

728

728

964

827

730

732

686

686

694

705

15KU

894812

161357

1507

1523

1474

1423

1350

1395

563

132

399

188

308

304

7646

493

506

472

472

617

542

486

494

515

517

529

529

16KU

894813

171211

1359

1390

1362

1366

1302

1275

363

311

390

432

247

263

290

292

319

220

952

952

715

701

699

703

682

681

691

711

17KU

894814

182065

45

47

46

68

68

51

1415

1039

1568

1499

1453

1227

1031

1140

1358

1247

232

230

250

235

242

246

290

287

297

294

18KM

1894191Ac

antham

oeba

sp

191208

1352

1400

1348

1345

1315

1279

352

300

386

428

235

264

278

284

318

101263

995

756

740

740

741

722

719

728

690

19JQ

2716891Hartm

annella

verm

iform

is

201222

1399

1449

1394

1390

1356

1318

352

305

381

428

235

264

283

284

318

101303

06

756

740

741

742

722

721

729

690

20KT

1856251Ve

rmam

oeba

verm

iform

is

211329

1267

1311

1272

1232

1197

1211

470

78367

253

234

275

07

04

53

295

1193

278

278

855

761

760

718

718

723

685

21AF2938951Echinamoeba

exun

dans

221318

1307

1352

1311

1307

1231

1242

462

145

364

358

252

290

161

149

171

315

1230

299

299

149

754

750

697

699

701

674

22AJ

4892611Echinamoeba

thermarum

231299

1447

1517

1401

1438

1399

1359

458

287

189

389

164

189

229

249

242

295

1311

278

276

241

250

970

764

780

773

750

23AY

3646351Nuclearia

patte

rson

i

241323

1460

1532

1413

1451

1388

1370

450

278

189

384

156

183

224

246

239

288

1321

271

269

238

252

25

769

785

780

758

24AF4

846871

Nuclearia

simplex

251368

1641

1706

1608

1625

1539

1545

536

349

319

427

00

103

293

284

314

284

1452

266

266

271

298

205

200

961

917

855

25AF4

112761Bo

domorph

aminim

a

261392

1768

1875

1724

1702

1632

1626

527

348

315

426

1699

291

283

308

281

1556

266

263

270

293

191

186

23

936

867

26EU

6471741|

Cercomon

adida

271342

1569

1629

1539

1522

1479

1455

506

311

326

423

60

103

271

268

288

261

1393

252

250

260

290

203

196

7258

872

27EU

7091401Cercozoasp

281245

1358

1360

1348

1364

1312

1282

507

348

320

447

80

02

314

279

322

268

1197

267

267

279

288

192

186

105

100

105

28AY

7488061Cercomon

asagilis

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728


FLA Free-living amoebaFLS Fish landing siteG Gravitational forceg GramsGAE Granulomatous amoebic encephalitisHIVAIDS Human immunodeficiency

virusacquired immune deficiencysyndrome

KCB Kazinga Channel bankKCM Kazinga Channel middleKLA KampalaL Litermg MilligramsmL MillilitersmM MillimolarNCBI National Center for Biotechnology

InformationNo NumberPA Protected areaPAM Primary amoebic

meningoencephalitispg Picogrampmol PicomolesPrev PrevalenceQEPA Queen Elizabeth Protected Arearpm Revolutions per minuteSG Specific gravityUNESCO United Nations Educational

Scientific and Cultural OrganizationUn-ID Amoeba Unidentified amoebaV Volts120583S Microseconds

Competing Interests

There are no competing interests

References

[1] A J Martinez and G S Visvesvara ldquoFree-living amphizoic andopportunistic amebasrdquo Brain Pathology vol 7 no 1 pp 583ndash598 1997

[2] WHO Combating Waterborne Disease at the HouseholdLevelInternational Network to Promote HouseholdWater Treat-ment and Safe Storage World Health Organization GenevaSwitzerland 2007

[3] CDC Domestic Water Sanitation and Hygiene Epidemiol-ogy Center for Disease Control Atlanta Ga USA 2015httpwwwcdcgovnceziddfwedwaterbornedomestichtml

[4] H Abedkhojasteh M Niyyati F Rahimi M Hei-Dari SFarnia andM Rezaeian ldquoFirst report of Hartmannella keratitisin a cosmetic soft contact lens wearer in Iranrdquo Iranian Journalof Parasitology vol 8 no 3 pp 481ndash485 2013

[5] C Sente J Erume I Naigaga et al ldquoOccurrence and geneticcharacterisation ofAcanthamoeba spp from environmental anddomestic water sources in Queen Elizabeth Protected AreaUgandardquo Parasites amp Vectors vol 9 article 127 2016

[6] J De Jonckheere ldquoNaegleria australiensis sp nov anotherpathogenic Naegleria from waterrdquo Protistologica vol 17 pp423ndash429 1981

[7] J FDe Jonckheere ldquoIsoenzymepatterns of pathogenic andnon-pathogenic Naegleria spp using agarose isoelectric focusingrdquoAnnales de Microbiologie vol 133 no 2 pp 319ndash342 1982

[8] J F De Jonckheere ldquoA century of research on the amoeboflag-ellate genus Naegleriardquo Acta Protozoologica vol 41 no 4 pp309ndash342 2002

[9] CDC ldquoAcanthamoeba keratitis associated with contact lensesmdashUnited Statesrdquo Morbidity and Mortality Weekly Report vol 35no 25 pp 405ndash408 1986

[10] V A Dunand S M Hammer R Rossi et al ldquoParasitic sinusitisand otitis in patients infected with human immunodeficiencyvirus report of five cases and reviewrdquo Clinical Infectious Dis-eases vol 25 no 2 pp 267ndash272 1997

[11] S Amir Acanthamoeba castellanii as a Host and Model toStudy Bacterial Virulence Karolinska UniversitetssjukhusetHuddinge Sweden 2009

[12] R Dey P S Hoffman and I J Glomski ldquoGermination andamplification of anthrax spores by soil-dwelling amoebasrdquoApplied and Environmental Microbiology vol 78 no 22 pp8075ndash8081 2012

[13] R Solgi M Niyyati A Haghighi and E N Mojarad ldquoOccur-rence of thermotolerant Hartmannella vermiformis and Naeg-leria spp in hot springs of Ardebil Province Northwest IranrdquoIranian Journal of Parasitology vol 7 no 2 pp 47ndash52 2012

[14] Uganda CholeraWHORegional Office for Africa 2012 httpwwwafrowhointenclusters-a-programmesdpcepidemic-a-pandemic-alert-and-response3601-uganda-cholera-situation-as-of-31-march-2012html

[15] G Ojore Uganda 2000 Children Die of Diarrhoea in AfricaDaily-WaterAid New Vision Publishing Kampala Uganda2012

[16] Uganda rural water services Inadequate quantity low qualitysatisfiedusers IRC 2014 httpwwwircwashorgnewsuganda-rural-water-services-inadequate-quantity-low-quality-satisfied-users

[17] WHO Regional Office for Africa Emergencies Preparednessand Response to Typhoid Fever Uganda 2015 httpwwwwhointcsrdon17-march-2015-ugandaen

[18] Naegleria Infection Medscape Drugs amp Diseases 2015 httpemedicinemedscapecomarticle223910-overviewa5

[19] G S Visvesvara H Moura and F L Schuster ldquoPathogenic andopportunistic free-living amoebae Acanthamoeba spp Bala-muthia mandrillaris Naegleria fowleri and Sappinia diploideardquoFEMS Immunology and Medical Microbiology vol 50 no 1 pp1ndash26 2007

[20] L J Stockman C J Wright G S Visvesvara B S Fields andM J Beach ldquoPrevalence of Acanthamoeba spp and other free-living amoebae in household water Ohio USAmdash1990ndash1992rdquoParasitology Research vol 108 no 3 pp 621ndash627 2011

[21] G S Visvesvara ldquoAmebic meningoencephalitides and keratitischallenges in diagnosis and treatmentrdquo Current Opinion inInfectious Diseases vol 23 no 6 pp 590ndash594 2010

[22] N Crum-Cianflone Acanthamoeba Naval Medical Center atSan Diego San Diego Calif USA 2015

[23] F Marciano-Cabral and G A Cabral ldquoAcanthamoeba spp asagents of disease in humansrdquoClinicalMicrobiology Reviews vol16 no 2 pp 273ndash307 2003

[24] J Lorenzo-Morales E Martınez-Carretero N Batista et alldquoEarly diagnosis of amoebic keratitis due to a mixed infectionwith Acanthamoeba and Hartmannellardquo Parasitology Researchvol 102 no 1 pp 167ndash169 2007


[25] Z Szenasi T Endo K Yagita and ENagy ldquoIsolation identifica-tion and increasing importance of lsquofree-livingrsquo amoebae causinghuman diseaserdquo Journal of Medical Microbiology vol 47 no 1pp 5ndash16 1998

[26] H Trabelsi F Dendana A Sellami et al ldquoPathogenic free-living amoebae epidemiology and clinical reviewrdquo PathologieBiologie vol 60 no 6 pp 399ndash405 2012

[27] Uganda Bureau of Statistics Estimated Population of Kampalain 2002 2010 amp 2011 Uganda Bureau of Statistics 2014

[28] F L Schuster and G S Visvesvara ldquoFree-living amoebae asopportunistic and non-opportunistic pathogens of humans andanimalsrdquo International Journal for Parasitology vol 34 no 9 pp1001ndash1027 2004

[29] M Pelandakis and P Pernin ldquoUse of multiplex PCR andPCR restriction enzyme analysis for detection and explorationof the variability in the free-living amoeba Naegleria in theenvironmentrdquoApplied and EnvironmentalMicrobiology vol 68no 4 pp 2061ndash2065 2002

[30] J M Schroeder G C Booton J Hay et al ldquoUse of subgenic 18Sribosomal DNA PCR and sequencing for genus and genotypeidentification of Acanthamoebae from humans with keratitisand from sewage sludgerdquo Journal of Clinical Microbiology vol39 no 5 pp 1903ndash1911 2001

[31] G C Booton D J Kelly Y-W Chu et al ldquo18S ribosomalDNA typing and tracking of Acanthamoeba species isolatesfrom corneal scrape specimens contact lenses lens cases andhomewater supplies ofAcanthamoeba keratitis patients inHongKongrdquo Journal of Clinical Microbiology vol 40 no 5 pp 1621ndash1625 2002

[32] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[33] P Bonilla-Lemus A S Caballero Villegas J Carmona Jimenezand A Lugo Vazquez ldquoOccurrence of free-living amoebae instreams of the Mexico Basinrdquo Experimental Parasitology vol145 supplement pp S28ndashS33 2014

[34] S Ghadar-Ghadr K Solhjoo M Norouz-Nejad R Rohi and SZia-Jahromi ldquoIsolation and identification of free living amoeba(Naegleria and Acanthamoeba) in Shiraz water resources bymorphological criteriardquo Pars of Jahrom University of MedicalSciences vol 10 no 3 pp 33ndash42 2012

[35] S Ghadar-Ghadr K Solhjoo M Norouz-Nejad R Rohi and SZia-Jahromi ldquoIsolation and identification of free living amoeba(Naegleria and Acanthamoeba) in Shiraz water resources bymorphological criteriardquo Journal of Jahrom University of MedicalSciences vol 10 no 3 pp 26ndash33 2012

[36] S Rodriguez-Zaragoza E Mayzlish and Y Steinberger ldquoVer-tical distribution of the free-living amoeba population in soilunder desert shrubs in the Negev Desert Israelrdquo Applied andEnvironmentalMicrobiology vol 71 no 4 pp 2053ndash2060 2005

[37] M Pruden J Falkinham K Williams H Wang K Martinsand W Rhoads ldquoRelationship between biodegradable organicmatter and pathogen concentrations in premise plumbingrdquoWater Research Foundation pp 58ndash90 2013

[38] A Rozej A Cydzik-Kwiatkowska B Kowalska and D Kowal-ski ldquoStructure and microbial diversity of biofilms on differentpipe materials of a model drinking water distribution systemsrdquoWorld Journal of Microbiology amp Biotechnology vol 31 no 1 pp37ndash47 2015

[39] M H Vodkin D K Howe G S Visvesvara and G LMcLaughlin ldquoIdentification ofAcanthamoeba at the generic and

specific levels using the polymerase chain reactionrdquoThe Journalof Protozoology vol 39 no 3 pp 378ndash385 1992

[40] M Crary Genetic Variability and Its Relationship to Acan-thamoeba Pathogenesis Molecular Genetics Ohio State Univer-sity Columbus Ohio USA 2012

[41] M S Torno Jr R Babapour A Gurevitch and M D WittldquoCutaneous acanthamoebiasis inAIDSrdquo Journal of theAmericanAcademy of Dermatology vol 42 no 2 part 2 pp 351ndash3542000

[42] J F De Jonckheere and S Brown ldquoThere is no evidence that thefree-living ameba Hartmannella is a human parasiterdquo ClinicalInfectious Diseases vol 26 no 3 p 773 1998

[43] P W Woodburn L Muhangi S Hillier et al ldquoRisk factors forhelminth malaria and HIV infection in pregnancy in EntebbeUgandardquo PLoS Neglected Tropical Diseases vol 3 no 6 articlee473 2009

[44] S Onichandran T Kumar C C Salibay et al ldquoWaterborneparasites a current status from the Philippinesrdquo Parasites ampVectors vol 7 no 1 article 244 2014

[45] T Kumar S Onichandran Y A L Lim et al ldquoComparativestudy on waterborne parasites between Malaysia andThailanda new insightrdquo The American Journal of Tropical Medicine andHygiene vol 90 no 4 pp 682ndash689 2014

[46] D CarmenaWaterborne Transmission of Cryptosporidium andGiardia Detection Surveillance and Implications for PublicHealth Faculty of Medicine IC London UK 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


A castellanii A culbertsoni A rhysodes A lugdunensisA quina A griffini Balamuthia mandrillaris Hartmannellavermiformis and Vahlkampfia avara as pathogenic speciesmostly associated with keratitis in humans [9 10] Acan-thamoeba spp particularly renders amplification of Vibriocholerae Legionella pneumophila Bacillus anthracis andMycoplasma tuberculosis inside their cells [10ndash12] a propertyof paramount importance to both humans and wildlife inand around QEPA Hartmannella spp that have in manyinstances been isolated in mixed human amoebic keratitisinfections are recently considered to be very pathogenic FLA[13] This work therefore reports the different pathogenicFLA associated with tap and environmental surface water inQEPA and Kampala an indicator towards water toxicity forthe communities In the past 10 years outbreaks of choleratyphoid and other waterborne diseases have been docu-mented in the study areas [14ndash17] and these could be linkedto distribution by contaminated water sources as well

The FLA of the genera Naegleria Acanthamoeba andHartmannella predispose humans who collect water fromvarious sources to diseases [1 10 18] These pathogenic andopportunistic FLA are aerobic eukaryotic protists that occurworldwide and can potentially cause infections in humansand other animals [10 19 20] The bacterial parasites Vibriocholerae Legionella pneumophila Mycoplasma tuberculosisand Bacillus anthracis are easily amplified within certainAcanthamoeba spp Hartmannella spp Naegleria spp andother FLA consequently increasing their potential to causecholera legionellosis tuberculosis and anthrax respectively[10ndash12 19 20]

The risks associated with FLA infection inUganda are notknown the reason why this study is being carried out Theassociations are expected to be high because significant por-tions of these communities are using natural environmentalwater and domestic tap water sources without any definitemeasures for protecting water systems in QEPA and KLAfrom these organisms [5] FLA are not often mentioned aspossible infectious agents yet they have severe pathogeniceffects [25 26] Disease effects associated with pathogenicFLA in humans and animals often go undetected becausethere is scarce information about their distribution InUganda the gap being addressed is that of studyingpathogenic FLA in environmental water and domestic tapwater systems This information will help explain the risksassociatedwithwater-related illnesses among rural and urbandwelling Ugandans

2 Methods

21 Study Areas The study was conducted in a rural areaQueen Elizabeth Protected Area (QEPA) in western Ugandaand an urban area Kampala (KLA) City Uganda TheQEPA is located at 00 12S 30 00E (latitude 02000 lon-gitude 300000) and is 1978 sq km in size The pro-tected area is known for its wildlife including Cape buf-faloes (Syncerus caffer caffer) Hippopotami (Hippopotamusamphibius) Nile Crocodiles (Crocodylus niloticus) elephants

(Loxodonta africana) leopards (Panthera pardus) lions (Pan-thera leo) chimpanzees (Pan troglodytes) and Uganda kobs(Kobus kob thomasi) It is a home to 95 species of mammalsand more than 500 species of birds The protected area isalso famous for its volcanic features including volcanic conesand deep craters QEPA is a UNESCO ldquoMan and BiosphereReserverdquo with 11 village enclaves all with a fast growing pop-ulation of humanswhosemain economic activities are fishingand livestock production The diversity of animals in theprotected area coupled with adjacent human communitiesmakes it a vital hub for sharing of infections at water pointsources

Kampala is the capital city of Uganda lying at latitude0∘1810158405810158401015840N longitude 32∘3410158405510158401015840E with 7297mi2 (189 km2)and divided into five boroughs each consisting of a con-centration of slums with a rapidly growing populationCurrently estimated at 1659600 [27] Kampalarsquos population isincreasing steadily yet service provision is not improvingWater resources used in the city vary from piped waterprotected springs unprotected springs and natural surfacewater (lakes rivers streams swamps roadside gutters andpasture puddles) which are often relatively unsafe for humanconsumption

22 Ethical Consideration This study does not require anethical statement

23 Study Design The study involved one year of field workand one year of confirmatory laboratory phase Laboratoryphase involved analysing water samples for FLA The watersources considered were those from natural environmentaland domestic tap water sources The sampling sites werepurposively selected based on their benefit convenience andimportance to public health

In QEPA selection of the study area was based on certainlandmarks that included the following (1) along the Kyam-buraRiver (2)KazingaChannel banks (3)KazingaMidchan-nel (4) fish landing sites (FLS) and (5) community pipedtap water In Kampala the location of sampling sites wasbased on previous waterborne disease outbreak occurrencesWe selected areas in central Kampala (Banda KisenyiKatanga Kasubi Kazo Bwaise Lubigi and Makerere) thatwere reported to have undergone disease outbreak in the last 5years and collected samples from piped tap water and naturalenvironmental surface water (swamp and stream)

24 Laboratory Methods

241 Xenic Cultivation of FLA from Water Samples Weused nonnutritive medium (Page Amoeba Saline solution of25mM NaCl 1mM KH

2PO4 05mM Na

2HPO4 40mM

CaCl2 and 20mM MgSO

4) seeded with 01mL of a heat

inactivated 48-hour culture of Escherichia coli BL2 [5 28]Fresh water samples in the 50mL tubes were centrifuged at1000timesg for 15 minutes and supernatant was poured off toexpose the pellets Using sterile Pasteur pipettes the pelletswere removed from all the tubes and each was carefullyspreadplated on preseeded NNA-EI agar plates This was







QEPA KLA



(n = 204) (n = 174)(n = 116)(n = 204)

010203040506070

Ove

rall

prev

alen

ce




3 Results



















4 Discussion



1

02

072

008

009

004

006

012

014

089

0861

0

10

078

087

077

091

077

091

018

058

063

054

088

033

085

KU894812


KU894808

KU894813

KU894806


KU894814


KU894799

KU894800

KU894801

KU894802

KU894804

















[22 23]











5 Conclusion




Table4Estim

ationof

evolutionary

divergence

betweensequ

ences

Percentidentity

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

Divergence

1532

536

534

528

538

538

514

521

565

345

360

315

522

256

533

254

537

252

251

248

243

250

252

305

299

305

309

1KU

884884

22391

973

956

960

944

944

533

554

549

329

346

307

554

264

525

239

915

238

235

256

242

246

246

293

289

299

310

2KU

894799

32203

19957

950

940

945

523

544

550

328

348

306

545

266

525

238

917

236

234

255

240

243

247

291

289

298

311

3KU

894800

42051

30

37

941

934

956

528

552

550

323

346

306

550

264

525

242

898

238

235

255

240

248

252

294

290

301

311

4KU

894801

52187

40

42

33

940

944

529

550

544

333

342

303

550

266

526

236

897

236

234

256

239

244

248

291

289

299

311

5KU

894802

62120

48

60

53

40

940

521

540

558

342

356

318

540

278

538

247

887

243

240

264

250

250

255

298

294

305

317

6KU

894803

72262

33

45

1743

57

509

530

554

332

353

315

530

275

536

251

890

246

243

263

248

254

258

299

298

307

326

7KU

894804

81310

1459

1409

1424

1447

1441

1388

795

632

357

370

329

807

277

546

283

562

277

277

256

256

256

264

302

299

305

310

8KU

894805

91271

1043

1030

1014

1032

1048

1006

484

659

455

420

362

960

430

666

322

583

323

322

416

381

315

322

356

353

364

352

9KU

894806

101335

1819

1777

1637

1793

1671

1584

500

389

474

537

491

670

415

666

389

581

381

383

395

395

479

486

471

477

482

485

10KU

894807

111410

1653

1636

1653

1490

1472

1564

640

264

483

652

596

478

677

651

553

313

545

545

654

585

549

556

588

591

592

589

11KU894808

121368

1691

1709

1633

1677

1562

1534

531

342

316

424

836

432

695

583

685

333

689

689

689

675

733

742

928

898

863

846

12KU

894809

131216

1391

1404

1392

1384

1332

1314

472

338

304

422

79374

701

534

697

291

683

683

681

664

744

752

847

860

864

958

13KU

894810

141279

1029

1026

1014

1017

1034

987

456

69

329

211

294

296

452

687

328

584

325

323

436

368

323

332

365

362

369

360

14KU

894811

151330

1238

1257

1247

1192

1156

1180

463

66

362

248

241

272

00

648

742

247

728

728

964

827

730

732

686

686

694

705

15KU

894812

161357

1507

1523

1474

1423

1350

1395

563

132

399

188

308

304

7646

493

506

472

472

617

542

486

494

515

517

529

529

16KU

894813

171211

1359

1390

1362

1366

1302

1275

363

311

390

432

247

263

290

292

319

220

952

952

715

701

699

703

682

681

691

711

17KU

894814

182065

45

47

46

68

68

51

1415

1039

1568

1499

1453

1227

1031

1140

1358

1247

232

230

250

235

242

246

290

287

297

294

18KM

1894191Ac

antham

oeba

sp

191208

1352

1400

1348

1345

1315

1279

352

300

386

428

235

264

278

284

318

101263

995

756

740

740

741

722

719

728

690

19JQ

2716891Hartm

annella

verm

iform

is

201222

1399

1449

1394

1390

1356

1318

352

305

381

428

235

264

283

284

318

101303

06

756

740

741

742

722

721

729

690

20KT

1856251Ve

rmam

oeba

verm

iform

is

211329

1267

1311

1272

1232

1197

1211

470

78367

253

234

275

07

04

53

295

1193

278

278

855

761

760

718

718

723

685


exun

dans

221318

1307

1352

1311

1307

1231

1242

462

145

364

358

252

290

161

149

171

315

1230

299

299

149

754

750

697

699

701

674

22AJ

4892611Echinamoeba

thermarum

231299

1447

1517

1401

1438

1399

1359

458

287

189

389

164

189

229

249

242

295

1311

278

276

241

250

970

764

780

773

750

23AY

3646351Nuclearia

patte

rson

i

241323

1460

1532

1413

1451

1388

1370

450

278

189

384

156

183

224

246

239

288

1321

271

269

238

252

25

769

785

780

758

24AF4

846871

Nuclearia

simplex

251368

1641

1706

1608

1625

1539

1545

536

349

319

427

00

103

293

284

314

284

1452

266

266

271

298

205

200

961

917

855

25AF4

112761Bo

domorph

aminim

a

261392

1768

1875

1724

1702

1632

1626

527

348

315

426

1699

291

283

308

281

1556

266

263

270

293

191

186

23

936

867

26EU

6471741|

Cercomon

adida

271342

1569

1629

1539

1522

1479

1455

506

311

326

423

60

103

271

268

288

261

1393

252

250

260

290

203

196

7258

872

27EU

7091401Cercozoasp

281245

1358

1360

1348

1364

1312

1282

507

348

320

447

80

02

314

279

322

268

1197

267

267

279

288

192

186

105

100

105

28AY

7488061Cercomon

asagilis

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728








Competing Interests


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







QEPA KLA



(n = 204) (n = 174)(n = 116)(n = 204)

010203040506070

Ove

rall

prev

alen

ce




3 Results



















4 Discussion



1

02

072

008

009

004

006

012

014

089

0861

0

10

078

087

077

091

077

091

018

058

063

054

088

033

085

KU894812


KU894808

KU894813

KU894806


KU894814


KU894799

KU894800

KU894801

KU894802

KU894804

















[22 23]











5 Conclusion




Table4Estim

ationof

evolutionary

divergence

betweensequ

ences

Percentidentity

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

Divergence

1532

536

534

528

538

538

514

521

565

345

360

315

522

256

533

254

537

252

251

248

243

250

252

305

299

305

309

1KU

884884

22391

973

956

960

944

944

533

554

549

329

346

307

554

264

525

239

915

238

235

256

242

246

246

293

289

299

310

2KU

894799

32203

19957

950

940

945

523

544

550

328

348

306

545

266

525

238

917

236

234

255

240

243

247

291

289

298

311

3KU

894800

42051

30

37

941

934

956

528

552

550

323

346

306

550

264

525

242

898

238

235

255

240

248

252

294

290

301

311

4KU

894801

52187

40

42

33

940

944

529

550

544

333

342

303

550

266

526

236

897

236

234

256

239

244

248

291

289

299

311

5KU

894802

62120

48

60

53

40

940

521

540

558

342

356

318

540

278

538

247

887

243

240

264

250

250

255

298

294

305

317

6KU

894803

72262

33

45

1743

57

509

530

554

332

353

315

530

275

536

251

890

246

243

263

248

254

258

299

298

307

326

7KU

894804

81310

1459

1409

1424

1447

1441

1388

795

632

357

370

329

807

277

546

283

562

277

277

256

256

256

264

302

299

305

310

8KU

894805

91271

1043

1030

1014

1032

1048

1006

484

659

455

420

362

960

430

666

322

583

323

322

416

381

315

322

356

353

364

352

9KU

894806

101335

1819

1777

1637

1793

1671

1584

500

389

474

537

491

670

415

666

389

581

381

383

395

395

479

486

471

477

482

485

10KU

894807

111410

1653

1636

1653

1490

1472

1564

640

264

483

652

596

478

677

651

553

313

545

545

654

585

549

556

588

591

592

589

11KU894808

121368

1691

1709

1633

1677

1562

1534

531

342

316

424

836

432

695

583

685

333

689

689

689

675

733

742

928

898

863

846

12KU

894809

131216

1391

1404

1392

1384

1332

1314

472

338

304

422

79374

701

534

697

291

683

683

681

664

744

752

847

860

864

958

13KU

894810

141279

1029

1026

1014

1017

1034

987

456

69

329

211

294

296

452

687

328

584

325

323

436

368

323

332

365

362

369

360

14KU

894811

151330

1238

1257

1247

1192

1156

1180

463

66

362

248

241

272

00

648

742

247

728

728

964

827

730

732

686

686

694

705

15KU

894812

161357

1507

1523

1474

1423

1350

1395

563

132

399

188

308

304

7646

493

506

472

472

617

542

486

494

515

517

529

529

16KU

894813

171211

1359

1390

1362

1366

1302

1275

363

311

390

432

247

263

290

292

319

220

952

952

715

701

699

703

682

681

691

711

17KU

894814

182065

45

47

46

68

68

51

1415

1039

1568

1499

1453

1227

1031

1140

1358

1247

232

230

250

235

242

246

290

287

297

294

18KM

1894191Ac

antham

oeba

sp

191208

1352

1400

1348

1345

1315

1279

352

300

386

428

235

264

278

284

318

101263

995

756

740

740

741

722

719

728

690

19JQ

2716891Hartm

annella

verm

iform

is

201222

1399

1449

1394

1390

1356

1318

352

305

381

428

235

264

283

284

318

101303

06

756

740

741

742

722

721

729

690

20KT

1856251Ve

rmam

oeba

verm

iform

is

211329

1267

1311

1272

1232

1197

1211

470

78367

253

234

275

07

04

53

295

1193

278

278

855

761

760

718

718

723

685


exun

dans

221318

1307

1352

1311

1307

1231

1242

462

145

364

358

252

290

161

149

171

315

1230

299

299

149

754

750

697

699

701

674

22AJ

4892611Echinamoeba

thermarum

231299

1447

1517

1401

1438

1399

1359

458

287

189

389

164

189

229

249

242

295

1311

278

276

241

250

970

764

780

773

750

23AY

3646351Nuclearia

patte

rson

i

241323

1460

1532

1413

1451

1388

1370

450

278

189

384

156

183

224

246

239

288

1321

271

269

238

252

25

769

785

780

758

24AF4

846871

Nuclearia

simplex

251368

1641

1706

1608

1625

1539

1545

536

349

319

427

00

103

293

284

314

284

1452

266

266

271

298

205

200

961

917

855

25AF4

112761Bo

domorph

aminim

a

261392

1768

1875

1724

1702

1632

1626

527

348

315

426

1699

291

283

308

281

1556

266

263

270

293

191

186

23

936

867

26EU

6471741|

Cercomon

adida

271342

1569

1629

1539

1522

1479

1455

506

311

326

423

60

103

271

268

288

261

1393

252

250

260

290

203

196

7258

872

27EU

7091401Cercozoasp

281245

1358

1360

1348

1364

1312

1282

507

348

320

447

80

02

314

279

322

268

1197

267

267

279

288

192

186

105

100

105

28AY

7488061Cercomon

asagilis

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728








Competing Interests


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















4 Discussion



1

02

072

008

009

004

006

012

014

089

0861

0

10

078

087

077

091

077

091

018

058

063

054

088

033

085

KU894812


KU894808

KU894813

KU894806


KU894814


KU894799

KU894800

KU894801

KU894802

KU894804

















[22 23]











5 Conclusion




Table4Estim

ationof

evolutionary

divergence

betweensequ

ences

Percentidentity

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

Divergence

1532

536

534

528

538

538

514

521

565

345

360

315

522

256

533

254

537

252

251

248

243

250

252

305

299

305

309

1KU

884884

22391

973

956

960

944

944

533

554

549

329

346

307

554

264

525

239

915

238

235

256

242

246

246

293

289

299

310

2KU

894799

32203

19957

950

940

945

523

544

550

328

348

306

545

266

525

238

917

236

234

255

240

243

247

291

289

298

311

3KU

894800

42051

30

37

941

934

956

528

552

550

323

346

306

550

264

525

242

898

238

235

255

240

248

252

294

290

301

311

4KU

894801

52187

40

42

33

940

944

529

550

544

333

342

303

550

266

526

236

897

236

234

256

239

244

248

291

289

299

311

5KU

894802

62120

48

60

53

40

940

521

540

558

342

356

318

540

278

538

247

887

243

240

264

250

250

255

298

294

305

317

6KU

894803

72262

33

45

1743

57

509

530

554

332

353

315

530

275

536

251

890

246

243

263

248

254

258

299

298

307

326

7KU

894804

81310

1459

1409

1424

1447

1441

1388

795

632

357

370

329

807

277

546

283

562

277

277

256

256

256

264

302

299

305

310

8KU

894805

91271

1043

1030

1014

1032

1048

1006

484

659

455

420

362

960

430

666

322

583

323

322

416

381

315

322

356

353

364

352

9KU

894806

101335

1819

1777

1637

1793

1671

1584

500

389

474

537

491

670

415

666

389

581

381

383

395

395

479

486

471

477

482

485

10KU

894807

111410

1653

1636

1653

1490

1472

1564

640

264

483

652

596

478

677

651

553

313

545

545

654

585

549

556

588

591

592

589

11KU894808

121368

1691

1709

1633

1677

1562

1534

531

342

316

424

836

432

695

583

685

333

689

689

689

675

733

742

928

898

863

846

12KU

894809

131216

1391

1404

1392

1384

1332

1314

472

338

304

422

79374

701

534

697

291

683

683

681

664

744

752

847

860

864

958

13KU

894810

141279

1029

1026

1014

1017

1034

987

456

69

329

211

294

296

452

687

328

584

325

323

436

368

323

332

365

362

369

360

14KU

894811

151330

1238

1257

1247

1192

1156

1180

463

66

362

248

241

272

00

648

742

247

728

728

964

827

730

732

686

686

694

705

15KU

894812

161357

1507

1523

1474

1423

1350

1395

563

132

399

188

308

304

7646

493

506

472

472

617

542

486

494

515

517

529

529

16KU

894813

171211

1359

1390

1362

1366

1302

1275

363

311

390

432

247

263

290

292

319

220

952

952

715

701

699

703

682

681

691

711

17KU

894814

182065

45

47

46

68

68

51

1415

1039

1568

1499

1453

1227

1031

1140

1358

1247

232

230

250

235

242

246

290

287

297

294

18KM

1894191Ac

antham

oeba

sp

191208

1352

1400

1348

1345

1315

1279

352

300

386

428

235

264

278

284

318

101263

995

756

740

740

741

722

719

728

690

19JQ

2716891Hartm

annella

verm

iform

is

201222

1399

1449

1394

1390

1356

1318

352

305

381

428

235

264

283

284

318

101303

06

756

740

741

742

722

721

729

690

20KT

1856251Ve

rmam

oeba

verm

iform

is

211329

1267

1311

1272

1232

1197

1211

470

78367

253

234

275

07

04

53

295

1193

278

278

855

761

760

718

718

723

685


exun

dans

221318

1307

1352

1311

1307

1231

1242

462

145

364

358

252

290

161

149

171

315

1230

299

299

149

754

750

697

699

701

674

22AJ

4892611Echinamoeba

thermarum

231299

1447

1517

1401

1438

1399

1359

458

287

189

389

164

189

229

249

242

295

1311

278

276

241

250

970

764

780

773

750

23AY

3646351Nuclearia

patte

rson

i

241323

1460

1532

1413

1451

1388

1370

450

278

189

384

156

183

224

246

239

288

1321

271

269

238

252

25

769

785

780

758

24AF4

846871

Nuclearia

simplex

251368

1641

1706

1608

1625

1539

1545

536

349

319

427

00

103

293

284

314

284

1452

266

266

271

298

205

200

961

917

855

25AF4

112761Bo

domorph

aminim

a

261392

1768

1875

1724

1702

1632

1626

527

348

315

426

1699

291

283

308

281

1556

266

263

270

293

191

186

23

936

867

26EU

6471741|

Cercomon

adida

271342

1569

1629

1539

1522

1479

1455

506

311

326

423

60

103

271

268

288

261

1393

252

250

260

290

203

196

7258

872

27EU

7091401Cercozoasp

281245

1358

1360

1348

1364

1312

1282

507

348

320

447

80

02

314

279

322

268

1197

267

267

279

288

192

186

105

100

105

28AY

7488061Cercomon

asagilis

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728








Competing Interests


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1

02

072

008

009

004

006

012

014

089

0861

0

10

078

087

077

091

077

091

018

058

063

054

088

033

085

KU894812


KU894808

KU894813

KU894806


KU894814


KU894799

KU894800

KU894801

KU894802

KU894804

















[22 23]











5 Conclusion




Table4Estim

ationof

evolutionary

divergence

betweensequ

ences

Percentidentity

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

Divergence

1532

536

534

528

538

538

514

521

565

345

360

315

522

256

533

254

537

252

251

248

243

250

252

305

299

305

309

1KU

884884

22391

973

956

960

944

944

533

554

549

329

346

307

554

264

525

239

915

238

235

256

242

246

246

293

289

299

310

2KU

894799

32203

19957

950

940

945

523

544

550

328

348

306

545

266

525

238

917

236

234

255

240

243

247

291

289

298

311

3KU

894800

42051

30

37

941

934

956

528

552

550

323

346

306

550

264

525

242

898

238

235

255

240

248

252

294

290

301

311

4KU

894801

52187

40

42

33

940

944

529

550

544

333

342

303

550

266

526

236

897

236

234

256

239

244

248

291

289

299

311

5KU

894802

62120

48

60

53

40

940

521

540

558

342

356

318

540

278

538

247

887

243

240

264

250

250

255

298

294

305

317

6KU

894803

72262

33

45

1743

57

509

530

554

332

353

315

530

275

536

251

890

246

243

263

248

254

258

299

298

307

326

7KU

894804

81310

1459

1409

1424

1447

1441

1388

795

632

357

370

329

807

277

546

283

562

277

277

256

256

256

264

302

299

305

310

8KU

894805

91271

1043

1030

1014

1032

1048

1006

484

659

455

420

362

960

430

666

322

583

323

322

416

381

315

322

356

353

364

352

9KU

894806

101335

1819

1777

1637

1793

1671

1584

500

389

474

537

491

670

415

666

389

581

381

383

395

395

479

486

471

477

482

485

10KU

894807

111410

1653

1636

1653

1490

1472

1564

640

264

483

652

596

478

677

651

553

313

545

545

654

585

549

556

588

591

592

589

11KU894808

121368

1691

1709

1633

1677

1562

1534

531

342

316

424

836

432

695

583

685

333

689

689

689

675

733

742

928

898

863

846

12KU

894809

131216

1391

1404

1392

1384

1332

1314

472

338

304

422

79374

701

534

697

291

683

683

681

664

744

752

847

860

864

958

13KU

894810

141279

1029

1026

1014

1017

1034

987

456

69

329

211

294

296

452

687

328

584

325

323

436

368

323

332

365

362

369

360

14KU

894811

151330

1238

1257

1247

1192

1156

1180

463

66

362

248

241

272

00

648

742

247

728

728

964

827

730

732

686

686

694

705

15KU

894812

161357

1507

1523

1474

1423

1350

1395

563

132

399

188

308

304

7646

493

506

472

472

617

542

486

494

515

517

529

529

16KU

894813

171211

1359

1390

1362

1366

1302

1275

363

311

390

432

247

263

290

292

319

220

952

952

715

701

699

703

682

681

691

711

17KU

894814

182065

45

47

46

68

68

51

1415

1039

1568

1499

1453

1227

1031

1140

1358

1247

232

230

250

235

242

246

290

287

297

294

18KM

1894191Ac

antham

oeba

sp

191208

1352

1400

1348

1345

1315

1279

352

300

386

428

235

264

278

284

318

101263

995

756

740

740

741

722

719

728

690

19JQ

2716891Hartm

annella

verm

iform

is

201222

1399

1449

1394

1390

1356

1318

352

305

381

428

235

264

283

284

318

101303

06

756

740

741

742

722

721

729

690

20KT

1856251Ve

rmam

oeba

verm

iform

is

211329

1267

1311

1272

1232

1197

1211

470

78367

253

234

275

07

04

53

295

1193

278

278

855

761

760

718

718

723

685


exun

dans

221318

1307

1352

1311

1307

1231

1242

462

145

364

358

252

290

161

149

171

315

1230

299

299

149

754

750

697

699

701

674

22AJ

4892611Echinamoeba

thermarum

231299

1447

1517

1401

1438

1399

1359

458

287

189

389

164

189

229

249

242

295

1311

278

276

241

250

970

764

780

773

750

23AY

3646351Nuclearia

patte

rson

i

241323

1460

1532

1413

1451

1388

1370

450

278

189

384

156

183

224

246

239

288

1321

271

269

238

252

25

769

785

780

758

24AF4

846871

Nuclearia

simplex

251368

1641

1706

1608

1625

1539

1545

536

349

319

427

00

103

293

284

314

284

1452

266

266

271

298

205

200

961

917

855

25AF4

112761Bo

domorph

aminim

a

261392

1768

1875

1724

1702

1632

1626

527

348

315

426

1699

291

283

308

281

1556

266

263

270

293

191

186

23

936

867

26EU

6471741|

Cercomon

adida

271342

1569

1629

1539

1522

1479

1455

506

311

326

423

60

103

271

268

288

261

1393

252

250

260

290

203

196

7258

872

27EU

7091401Cercozoasp

281245

1358

1360

1348

1364

1312

1282

507

348

320

447

80

02

314

279

322

268

1197

267

267

279

288

192

186

105

100

105

28AY

7488061Cercomon

asagilis

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728








Competing Interests


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








[22 23]











5 Conclusion




Table4Estim

ationof

evolutionary

divergence

betweensequ

ences

Percentidentity

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

Divergence

1532

536

534

528

538

538

514

521

565

345

360

315

522

256

533

254

537

252

251

248

243

250

252

305

299

305

309

1KU

884884

22391

973

956

960

944

944

533

554

549

329

346

307

554

264

525

239

915

238

235

256

242

246

246

293

289

299

310

2KU

894799

32203

19957

950

940

945

523

544

550

328

348

306

545

266

525

238

917

236

234

255

240

243

247

291

289

298

311

3KU

894800

42051

30

37

941

934

956

528

552

550

323

346

306

550

264

525

242

898

238

235

255

240

248

252

294

290

301

311

4KU

894801

52187

40

42

33

940

944

529

550

544

333

342

303

550

266

526

236

897

236

234

256

239

244

248

291

289

299

311

5KU

894802

62120

48

60

53

40

940

521

540

558

342

356

318

540

278

538

247

887

243

240

264

250

250

255

298

294

305

317

6KU

894803

72262

33

45

1743

57

509

530

554

332

353

315

530

275

536

251

890

246

243

263

248

254

258

299

298

307

326

7KU

894804

81310

1459

1409

1424

1447

1441

1388

795

632

357

370

329

807

277

546

283

562

277

277

256

256

256

264

302

299

305

310

8KU

894805

91271

1043

1030

1014

1032

1048

1006

484

659

455

420

362

960

430

666

322

583

323

322

416

381

315

322

356

353

364

352

9KU

894806

101335

1819

1777

1637

1793

1671

1584

500

389

474

537

491

670

415

666

389

581

381

383

395

395

479

486

471

477

482

485

10KU

894807

111410

1653

1636

1653

1490

1472

1564

640

264

483

652

596

478

677

651

553

313

545

545

654

585

549

556

588

591

592

589

11KU894808

121368

1691

1709

1633

1677

1562

1534

531

342

316

424

836

432

695

583

685

333

689

689

689

675

733

742

928

898

863

846

12KU

894809

131216

1391

1404

1392

1384

1332

1314

472

338

304

422

79374

701

534

697

291

683

683

681

664

744

752

847

860

864

958

13KU

894810

141279

1029

1026

1014

1017

1034

987

456

69

329

211

294

296

452

687

328

584

325

323

436

368

323

332

365

362

369

360

14KU

894811

151330

1238

1257

1247

1192

1156

1180

463

66

362

248

241

272

00

648

742

247

728

728

964

827

730

732

686

686

694

705

15KU

894812

161357

1507

1523

1474

1423

1350

1395

563

132

399

188

308

304

7646

493

506

472

472

617

542

486

494

515

517

529

529

16KU

894813

171211

1359

1390

1362

1366

1302

1275

363

311

390

432

247

263

290

292

319

220

952

952

715

701

699

703

682

681

691

711

17KU

894814

182065

45

47

46

68

68

51

1415

1039

1568

1499

1453

1227

1031

1140

1358

1247

232

230

250

235

242

246

290

287

297

294

18KM

1894191Ac

antham

oeba

sp

191208

1352

1400

1348

1345

1315

1279

352

300

386

428

235

264

278

284

318

101263

995

756

740

740

741

722

719

728

690

19JQ

2716891Hartm

annella

verm

iform

is

201222

1399

1449

1394

1390

1356

1318

352

305

381

428

235

264

283

284

318

101303

06

756

740

741

742

722

721

729

690

20KT

1856251Ve

rmam

oeba

verm

iform

is

211329

1267

1311

1272

1232

1197

1211

470

78367

253

234

275

07

04

53

295

1193

278

278

855

761

760

718

718

723

685


exun

dans

221318

1307

1352

1311

1307

1231

1242

462

145

364

358

252

290

161

149

171

315

1230

299

299

149

754

750

697

699

701

674

22AJ

4892611Echinamoeba

thermarum

231299

1447

1517

1401

1438

1399

1359

458

287

189

389

164

189

229

249

242

295

1311

278

276

241

250

970

764

780

773

750

23AY

3646351Nuclearia

patte

rson

i

241323

1460

1532

1413

1451

1388

1370

450

278

189

384

156

183

224

246

239

288

1321

271

269

238

252

25

769

785

780

758

24AF4

846871

Nuclearia

simplex

251368

1641

1706

1608

1625

1539

1545

536

349

319

427

00

103

293

284

314

284

1452

266

266

271

298

205

200

961

917

855

25AF4

112761Bo

domorph

aminim

a

261392

1768

1875

1724

1702

1632

1626

527

348

315

426

1699

291

283

308

281

1556

266

263

270

293

191

186

23

936

867

26EU

6471741|

Cercomon

adida

271342

1569

1629

1539

1522

1479

1455

506

311

326

423

60

103

271

268

288

261

1393

252

250

260

290

203

196

7258

872

27EU

7091401Cercozoasp

281245

1358

1360

1348

1364

1312

1282

507

348

320

447

80

02

314

279

322

268

1197

267

267

279

288

192

186

105

100

105

28AY

7488061Cercomon

asagilis

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728








Competing Interests


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table4Estim

ationof

evolutionary

divergence

betweensequ

ences

Percentidentity

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

Divergence

1532

536

534

528

538

538

514

521

565

345

360

315

522

256

533

254

537

252

251

248

243

250

252

305

299

305

309

1KU

884884

22391

973

956

960

944

944

533

554

549

329

346

307

554

264

525

239

915

238

235

256

242

246

246

293

289

299

310

2KU

894799

32203

19957

950

940

945

523

544

550

328

348

306

545

266

525

238

917

236

234

255

240

243

247

291

289

298

311

3KU

894800

42051

30

37

941

934

956

528

552

550

323

346

306

550

264

525

242

898

238

235

255

240

248

252

294

290

301

311

4KU

894801

52187

40

42

33

940

944

529

550

544

333

342

303

550

266

526

236

897

236

234

256

239

244

248

291

289

299

311

5KU

894802

62120

48

60

53

40

940

521

540

558

342

356

318

540

278

538

247

887

243

240

264

250

250

255

298

294

305

317

6KU

894803

72262

33

45

1743

57

509

530

554

332

353

315

530

275

536

251

890

246

243

263

248

254

258

299

298

307

326

7KU

894804

81310

1459

1409

1424

1447

1441

1388

795

632

357

370

329

807

277

546

283

562

277

277

256

256

256

264

302

299

305

310

8KU

894805

91271

1043

1030

1014

1032

1048

1006

484

659

455

420

362

960

430

666

322

583

323

322

416

381

315

322

356

353

364

352

9KU

894806

101335

1819

1777

1637

1793

1671

1584

500

389

474

537

491

670

415

666

389

581

381

383

395

395

479

486

471

477

482

485

10KU

894807

111410

1653

1636

1653

1490

1472

1564

640

264

483

652

596

478

677

651

553

313

545

545

654

585

549

556

588

591

592

589

11KU894808

121368

1691

1709

1633

1677

1562

1534

531

342

316

424

836

432

695

583

685

333

689

689

689

675

733

742

928

898

863

846

12KU

894809

131216

1391

1404

1392

1384

1332

1314

472

338

304

422

79374

701

534

697

291

683

683

681

664

744

752

847

860

864

958

13KU

894810

141279

1029

1026

1014

1017

1034

987

456

69

329

211

294

296

452

687

328

584

325

323

436

368

323

332

365

362

369

360

14KU

894811

151330

1238

1257

1247

1192

1156

1180

463

66

362

248

241

272

00

648

742

247

728

728

964

827

730

732

686

686

694

705

15KU

894812

161357

1507

1523

1474

1423

1350

1395

563

132

399

188

308

304

7646

493

506

472

472

617

542

486

494

515

517

529

529

16KU

894813

171211

1359

1390

1362

1366

1302

1275

363

311

390

432

247

263

290

292

319

220

952

952

715

701

699

703

682

681

691

711

17KU

894814

182065

45

47

46

68

68

51

1415

1039

1568

1499

1453

1227

1031

1140

1358

1247

232

230

250

235

242

246

290

287

297

294

18KM

1894191Ac

antham

oeba

sp

191208

1352

1400

1348

1345

1315

1279

352

300

386

428

235

264

278

284

318

101263

995

756

740

740

741

722

719

728

690

19JQ

2716891Hartm

annella

verm

iform

is

201222

1399

1449

1394

1390

1356

1318

352

305

381

428

235

264

283

284

318

101303

06

756

740

741

742

722

721

729

690

20KT

1856251Ve

rmam

oeba

verm

iform

is

211329

1267

1311

1272

1232

1197

1211

470

78367

253

234

275

07

04

53

295

1193

278

278

855

761

760

718

718

723

685


exun

dans

221318

1307

1352

1311

1307

1231

1242

462

145

364

358

252

290

161

149

171

315

1230

299

299

149

754

750

697

699

701

674

22AJ

4892611Echinamoeba

thermarum

231299

1447

1517

1401

1438

1399

1359

458

287

189

389

164

189

229

249

242

295

1311

278

276

241

250

970

764

780

773

750

23AY

3646351Nuclearia

patte

rson

i

241323

1460

1532

1413

1451

1388

1370

450

278

189

384

156

183

224

246

239

288

1321

271

269

238

252

25

769

785

780

758

24AF4

846871

Nuclearia

simplex

251368

1641

1706

1608

1625

1539

1545

536

349

319

427

00

103

293

284

314

284

1452

266

266

271

298

205

200

961

917

855

25AF4

112761Bo

domorph

aminim

a

261392

1768

1875

1724

1702

1632

1626

527

348

315

426

1699

291

283

308

281

1556

266

263

270

293

191

186

23

936

867

26EU

6471741|

Cercomon

adida

271342

1569

1629

1539

1522

1479

1455

506

311

326

423

60

103

271

268

288

261

1393

252

250

260

290

203

196

7258

872

27EU

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Competing Interests


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Competing Interests


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article xenic cultivation and genotyping of

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