research article prevalence of protozoa species in

Research ArticlePrevalence of Protozoa Species in Drinking andEnvironmental Water Sources in Sudan

Salah Shanan12 Hadi Abd1 Magdi Bayoumi2 Amir Saeed1 and Gunnar Sandstroumlm1

1Division of Clinical Microbiology Department of Laboratory Medicine Karolinska Institute Karolinska University HospitalHuddinge 141 86 Stockholm Sweden2Faculty of Medical Laboratory Sciences University of Medical Sciences and Technology PO Box 12810 Khartoum Sudan

Correspondence should be addressed to Gunnar Sandstrom gunnarsandstromkise

Received 8 December 2014 Revised 30 January 2015 Accepted 2 February 2015

Academic Editor Kevin Tyler

Copyright copy 2015 Salah Shanan 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

Protozoa are eukaryotic cells distributed worldwide in nature and are receiving increasing attention as reservoirs and potentialvectors for the transmission of pathogenic bacteria In the environment on the other hand many genera of the protozoa are humanand animal pathogens Only limited information is available on these organisms in developing countries and so far no informationon their presence is available from Sudan It is necessary to establish a molecular identification of species of the protozoa fromdrinking and environmental water 600 water samples were collected from five states (Gadarif Khartoum Kordofan Juba andWad Madani) in Sudan and analysed by polymerase chain reaction (PCR) and sequencing 57 out of 600 water samples were PCRpositive for protozoa 38 out of the 57 positive samples were identified by sequencing to contain 66 protozoa species including19 (288) amoebae 17 (257) Apicomplexa 25 (379) ciliates and 5 (76) flagellates This study utilized molecular methodsidentified species belonging to all phyla of protozoa and presented a fast and accurate molecular detection and identification ofpathogenic as well as free-living protozoa in water uncovering hazards facing public health

1 Introduction

Protozoa are eukaryotic cells distributed worldwide in natureand are receiving increasing attention as human and animalpathogens and potential vehicles for the transmission of bac-teria in the environment Unfortunately one of two personsin the world is affected by waterborne or foodborne parasites[1]Theprotozoa are among themost commonparasitic path-ogens present in environmental samples They have multi-stage life cycles consisting of an active trophozoite stage and aresistant stage (oocyst or cyst) excreted in feces that is capableof infecting new hosts [2]

The protozoa are subdivided into four phyla dependingon their methods of locomotion Mastigophora (flagellates)move by using one or more flagella Sarcodina (amoebae)have extensions of the cytoplasm called pseudopodia assist-ing phagocytosis and motion in the organisms Ciliophora(ciliates) move by means of cilia Sporozoa (Apicomplexa)have no locomotion [3]

Over the past few decades pathogenic enteric proto-zoa have been increasingly implicated in compromising

the health of millions of people mostly in developing coun-triesThese protozoa contribute significantly to the staggeringcaseload of diarrheal disease morbidity encountered in theseregions and are also significant concerns in industrializedcountries despite improved sanitation [4]

Acanthamoeba is a genus of free-living amoebae (FLA)which are environmental eukaryotic cells distributed world-wide in nature [5 6] Life cycle of Acanthamoeba includestwo stages a feeding trophozoite and a dormant cyst stageAcanthamoeba supports bacterial growth and survival andsaves the bacteria from chlorination [7ndash9] increasing the riskof human illness caused by bacteria or Acanthamoeba Acan-thamoeba species are showing an increased role as humanpathogens causing encephalitis keratitis pneumonitis anddermatitis [10 11] over the globe and the infection routes aremostly from the environment

Cryptosporidium species and Giardia intestinalis aremajor pathogens in thewaterborne transmission of infectionsand they are able to persist in the environment due to therobustness of the oocysts and cysts The occurrence ofthese microorganisms in different types of water has been

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 345619 5 pageshttpdxdoiorg1011552015345619

2 BioMed Research International

Table 1 Number of positive samples by PCR utilizing primertargeting protozoal 18S rRNA gene

Location Positive samples Negative samples Total PrevalenceGadarif 19 114 133 143Juba 12 74 86 140Khartoum 6 60 66 91Kordofan 14 86 100 140Wad Madani 6 209 215 28Total 57 543 600 95

confirmed and a considerable number of waterborne out-breaks have been reported worldwide [12ndash14]

In Sudan Cryptosporidium species is an important causeof diarrhea in children and it is suggested that intrafamilialspread occurs [15] The highest prevalence of diarrhealdiseases was recorded among Port Sudan children (155)followed by children living in areas where people draw waterfrom unrectified hafirs (135) and finally children living inareas where water is drawn from rectified hafirs (60) [16]However there is no information about the presence of theprotozoal species except Cryptosporidium The aims of thisstudy were to detect protozoa from water samples by poly-merase chain reaction (PCR) using primer (P-SSU-342f andMedlin B) and identify species of the protozoa by sequencing

2 Material and Methods

21 SampleCollection 600 sampleswere collected fromwatersources (zeer hafir tank lake and stream) that belonged todifferent areas (Gadarif Khartoum Kordofan Juba andWadMadani) in Sudan for a one-year period in 2008 (Table 1Figure 1)

The hafir (an underground water reservoir designed forstoring rain water carried by streams and used for domesticwater supply and for agricultural purposes in rural areasin the Sudan) [17] is one of the most common sources ofwater in peripheral areas and different types of materials(contaminants) accumulate in it (due to erosion) as well asthe feces from wild and domestic animals The zeer is a tra-ditional Sudanese storage jug made of baked clay commonlyused to keep drinking water Water samples were collectedinto sterile 50mL tubes and transported to the laboratoryat Microbiology Department University of Medical Sciencesand Technology Khartoum

22 DNA Extraction The samples were centrifuged for10min at 4000 rpm The supernatant was removed and thesediment was used for DNA extraction The DNA wasextracted using routine proteinase K procedures using Qia-gen DNA mini kit (Qiagen Valencia CA USA)

23 DNA Amplification A protozoa-specific forward primer(P-SSU-342f) and Eukarya-specific reverse primer (MedlinB) targeting 18S rRNA gene were used The forward primerwas P-SSU-342f (51015840 CTTTCGATGGTAGTGTATTGGACT-AC-31015840) and the reverse primer was Medlin B (51015840-TGATCC-TTCTGCAGGTTCACCTAC-31015840) [18 19] in PCR to obtain

Figure 1 White color with arrows showing the surveyed areas inSudan and South Sudan

a product of about 1360 bp The PCR reaction performed at95∘C for 10min 35 cycles at 94∘C for 1min 50∘C for 1min72∘C for 1min final extension at 72∘C for 10min using 04120583Mof each primer The PCR amplification step was carried outand the final PCR reaction mixture was divided into twoparts One part was used for 2 agarose gel electrophoresiswith ethidium bromide staining and the other part was usedfor DNA sequencing

A castellanii was used as a positive control Detectionsensitivity was performed by serial dilution of A castellanii(10 cellsmL to 106 cellsmL)

24 Sequencing After samples collection DNA extractionand PCR analysis direct sequencing of the protozoa 18SrRNA gene was performed using purified nested PCR prod-ucts The sequencing was carried out by MWG operon(MWG operon Germany) The nucleotide sequences werethen edited and aligned using Blast program the sequenc-ing confirmed protozoal species according to the GenBankaccession numbers for their nucleotide sequences

25 Statistical Analysis 1205942 test was used to verify differencesin the existence of protozoa in water samples collected fromdifferent sources A 119875 value of le005 was considered statisti-cally significant

3 Results

The current study collected environmental water samplesand extracted amplified visualised and sequenced the PCRproducts using Blast program A detection limit of 10 cellswas found forAcanthamoeba according to the serial dilutions(Figure 2) The PCR amplification of the genomic DNAextracted from water samples produced a single band ofthe expected size of 1360 bp (Figure 3) Out of 600 watersamples 57 positive samples were detected by PCR and gel

BioMed Research International 3

M 101

102

103

104

105

106

1500bp

Figure 2 Detection sensitivity test of PCR of amoeba 18S rDNA

1 2 3 4 5 6 M

3000

1500

1000

500

400

300

Figure 3 Representative agarose gel (2) electrophoresis of PCRproducts of protozoal 18S rRNA Lanes 1 to 4 samples containprotozoa 5 and 6 are negative and positive controls (approximately1360 bp) M is molecular mass markers (1 bp)

electrophoresis utilizing primer targeting 18S rRNA gene ofthe protozoa (Table 1)

The PCR positive samples were sequenced 38 samplespassed sequencing and 19 samples failed to pass the sequenc-ing The sequenced samples yield 66 species of protozoawhich belonged to four phyla which were amoebae 288Apicomplexa 257 ciliates 379 and flagellates 76

The identified amoebae were Acanthamoeba castellaniiCapsaspora owczarzaki Dictyostelium purpureum Enta-moeba dispar and Naegleria gruberi The Apicomplexa wereAscogregarina taiwanensis Blastocystis hominis Cryptospo-ridium muris Cryptosporidium parvum Neospora caninumTheileria parva and Toxoplasma gondii The ciliates wereIchthyophthirius multifiliis Oxytricha trifallax and Stylony-chia lemnae The flagellates were Perkinsus marinus andTrichomonas vaginalis (Table 2)

4 Discussion

Unsafe water and poor sanitation and hygiene have beenreported to rank third among the 20 leading risk factors forhealth burden in developing countries including Sudan [20]

Hafir tank zeer lake and stream are the main sources ofdrinking water in Sudan especially in the rural areas and notonly used for humans Consumption is also used for animalsand accordingly this is the common source of contaminationwith protozoa and other harmful microorganisms Although

there is much scientific literature available concerning theassociation of protozoa with waterborne diseases this is notthe case in Sudan

Our study has pioneered the use of a PCR-based molec-ular test to search for potentially disease causing protozoain drinking water resulting in the detection of four phylaof well-documented pathogenic protozoa This is the firststudy in Sudan investigating the detection and identificationof protozoa found in drinking water The result will providevital information regarding the protozoa found at differentgeographic locations thus facilitating correlation of the iden-tified organisms with the clinical phenotypes of infectiousdisease prevalent among the population inhibiting specificgeographical locations

The result in this study demonstrated that protozoa arecommon in all water sources especially in Gadarif Kord-ofan and Juba and distributed even in all drinking watersources hafirs zeers lakes tanks and streams 66 speciesof protozoa were identified by sequencing and they includeamoebae species (288) Apicomplexa species (257) cili-ates (379) and flagellates (76)

In this context studies by Leiva et al [21] and Tsvetkovaet al [22] reported that the amoebae were found in 43 and61 respectively of the water samples Moreover in a recentstudy in Turkey only three species of free-living amoeba Acastellanii A polyphaga andHartmannella vermiformis wereidentified from tap water [23] In addition CryptosporidiumGiardia and Acanthamoeba were isolated from stations ofrecreational lakes in Malaysia [24]

However these studies detected a limited number ofamoebae Cryptosporidium and Giardia only in water sam-ples compared to our study that identified 49microorganismscomprising 16 species belonging to four different phyla ofprotozoa which gives an alert about the risk of water contam-ination since most of these protozoa are human pathogens orzoonotic parasites

To our knowledge no proper studies were performedin Sudan searching for protozoa in water sources HoweverAdam et al [15] reported thatCryptosporidiumwas an impor-tant cause of diarrhea in children in Sudan Most of the inter-national studies in the field detected limited numbers ofprotozoal species such as Cryptosporidium and Giardia [2526]

It is well known that a complex interaction betweenorganisms is found in aquatic environment especially amoe-bae and bacteria Free-living amoebae are eukaryotic cellsfound in nature and including several genera such as Acan-thamoeba Balamuthia Naegleria and Sappinia It has beenshown that Acanthamoebae benefits from E coli and Kleb-siella aerogenes as food In contrast the role of Acanthamoe-bae as hosts and vectors for bacteria has been proposed formany pathogenic bacteria [27]

The interaction between bacteria and amoebae is verycomplex Output of that interaction is depending on whetherthe interacted bacterium is extracellular or intracellular and ifit possesses a type three secretion system (TTSS) or not sinceTTSS effector proteins are observed to affect strongly outputof the interaction [27] Extracellular bacteria cannot multiplyinside amoeba cell andTTSS possessing extracellular bacteria


Table 2 Identified protozoa species by sequencing found in water samples collected from different sources and locations

Location Source Number of identified protozoaAmoebae Number Apicomplexa Number Ciliates Number Flagellates Number

Gadarif

Zeer A castellanii 2 T gondii 1 0 0

HafirC owczarzaki 1

C murisN caninum

11

I multifiliis 1P marinus 2D purpureum 1 O trifallax 4

A castellanii 2 S lemnae 3

Tank A castellanii 1 C muris 2 S lemnae 2 0E dispar 1 T parva 1 O trifallax 2

JubaLake A castellanii 3

C muris 2S lemnaeO trifallax

13 0N caninum 1

A taiwanensis 1

Zeer C owczarzaki 1 B hominis 1 O trifallax 1 0A castellanii 1

Khartoum Lake A castellanii 1 T gondii 1 O trifallax 2 0Zeer 0 0 C muris 1 0 0 T vaginitis 1

Kordofan HafirD purpureum 1

T gondiiA taiwanensis

21

O trifallaxS lemnae

42 P marinus 2N gruberi 1

E dispar 1

Wad Madani Stream A castellanii 1 C parvum 1 0 0 0C owczarzaki 1

Total 19 17 25 5

such as P aeruginosa kill the amoebae However the extracel-lular bacterium E coli does not possess TTSS and therefore itis ingested as food by the amoebae

An intracellular bacterium multiplies inside amoeba cellThe intracellular bacterium that does not possess TTSSF tularensis multiplies symbiotically inside the amoebaewhile TTSS possessing intracellular bacterium E coli K1 andShigella flexneri lyse the amoebae according to activation ofTTSS [27]

Facultative intracellular bacteria such as Vibrio choleraeare able to multiply in water and inside the environmentalphagocytic eukaryotes such as free-living amoebae There-fore presence of the amoebae in water will enhance presenceof bacteria such as V cholerae [7 28 29] Surprisingly in thiscontext Acanthamoeba species and V cholerae were detectedfrom the same water samples collected from different watersources in Sudan [27 30] and Newsome et al isolated anamoeba naturally harboring a distinctive Legionella species[31 32] V cholerae and Legionella pneumophila are wellknown as causative agents for thewaterborne diseases choleraand legionellosis

The protozoa have a double infectious role in publichealth They can cause infections by themselves such asamoebiasis cryptosporidiosis giardiasis amoebic encephali-tis and amoebic keratitis and they can act as a vector to theirintracellular bacteria to cause multiple infections of protozoaand bacteria [31]

In addition to our findings about the identified protozoain this paper Fletcher et al [33] reviewed that G intestinalisCryptosporidium spp and Entamoeba spp were the mostcommonly reported protozoa associated with enteric infec-tions and were associated mainly with food- and waterborneoutbreaks These enteric protozoa are isolated frequently

from diarrheal patients in developing regions such as Asiaand Sub-Saharan Africa [33] However recently Muchesaet al detected 128 Acanthamoeba from 172 wastewatersamples [34] Our study presents the first report aboutdetection of the following protozoaA castellanii T gondii Cowczarzaki B hominis C muris and T vaginitis from zeersfound in Gadarif Juba and Khartoum

Overall prevalence of detected protozoa by PCRwas 95and the prevalence in Gadarif Juba Khartoum Kordofanand Wad Madani was 143 140 91 140 and 28respectivelyThe prevalence of identified protozoa inGadarifJuba Khartoum and Kordofan was not statistically signif-icant by 1205942 test (119875 gt 005) Moreover the prevalence ofidentified protozoa in Wad Madani compared to those ofGadarif Juba and Kordofan was highly statistically signif-icant (119875 values were 00012 00007 and 00003 resp) Butthe prevalence of identified protozoa in Wad Madani andKhartoumwas not statistically significant119875 value was 01210

This result uncovers the danger coming from water tospread infections between populations and our study pre-sented a fast and accurate molecular detection and diagnosisof protozoa species in water

Conflict of Interests

The authors declare that they have no conflict of interests

References

[1] C N LMacpherson ldquoHuman behaviour and the epidemiologyof parasitic zoonosesrdquo International Journal for Parasitology vol35 no 11-12 pp 1319ndash1331 2005


[2] D S Zarlenga and J M Trout ldquoConcentrating purifying anddetecting waterborne parasitesrdquo Veterinary Parasitology vol126 no 1-2 pp 195ndash217 2004

[3] S K Soni Microbes A Source of Energy for 21st Century NewIndia Publishing Agency 2007

[4] J AMoss and R A Snyder ldquoPathogenic Protozoardquo inMicrobialSource Tracking Methods Applications and Case Studies CHagedorn A R Blanch and V J Harwood Eds pp 157ndash188Springer New York NY USA 2011

[5] T J Brown R T M Cursons and E A Keys ldquoAmoebaefrom antarctic soil and waterrdquo Applied and EnvironmentalMicrobiology vol 44 no 2 pp 491ndash493 1982

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

[7] H Abd A Saeed A Weintraub G B Nair and G SandstromldquoVibrio cholerae O1 strains are facultative intracellular bacteriaable to survive and multiply symbiotically inside the aquaticfree-living amoeba Acanthamoeba castellaniirdquo FEMS Microbi-ology Ecology vol 60 no 1 pp 33ndash39 2007

[8] H J Jeong E S Jang B I Han et al ldquoAcanthamoeba could it bean environmental host of Shigellardquo Experimental Parasitologyvol 115 no 2 pp 181ndash186 2007

[9] C H King E B Shotts Jr R E Wooley and K G PorterldquoSurvival of coliforms and bacterial pathogens within protozoaduring chlorinationrdquo Applied and Environmental Microbiologyvol 54 no 12 pp 3023ndash3033 1988

[10] AM Imam and SMahgoub ldquoBlindness due toAcanthamoebafirst case report from Sudanrdquo International Journal of HealthSciences (Qassim) vol 2 no 2 pp 163ndash166 2008

[11] R Walia J G Montoya G S Visvesvera G C Booton andR L Doyle ldquoA case of successful treatment of cutaneous Acan-thamoeba infection in a lung transplant recipientrdquo TransplantInfectious Disease vol 9 no 1 pp 51ndash54 2007

[12] P Karanis C Kourenti and H Smith ldquoWaterborne transmis-sion of protozoan parasites a worldwide review of outbreaksand lessons learntrdquo Journal of Water and Health vol 5 no 1 pp1ndash38 2007

[13] S Baldursson and P Karanis ldquoWaterborne transmission ofprotozoan parasites review of worldwide outbreaksmdashan update2004ndash2010rdquoWater Research vol 45 no 20 pp 6603ndash6614 2011

[14] C Mons A Dumetre S Gosselin C Galliot and L MoulinldquoMonitoring of Cryptosporidium and Giardia river contamina-tion in Paris areardquo Water Research vol 43 no 1 pp 211ndash2172009

[15] A A Adam H S Hassan P Shears and E Elshibly ldquoCryp-tosporidium in Khartoum Sudanrdquo East African Medical Jour-nal vol 71 no 11 pp 745ndash746 1994

[16] M A Awad El Karim B M El Hassan and K K HusseinldquoSocial and public health implication of water supply in aridzones in the Sudanrdquo Social Science and Medicine vol 20 no4 pp 393ndash398 1985

[17] A A E Gaddal ldquoRainwater catchment for future generationsrdquoin Proceedings of the 5th International Conference on RainWaterCistern Systems Keelung Taiwan 1991

[18] L Medlin H J Elwood S Stickel and M L Sogin ldquoThecharacterization of enzymatically amplified eukaryotic 16S-likerRNA-coding regionsrdquo Gene vol 71 no 2 pp 491ndash499 1988

[19] S K R Karnati Z Yu J T Sylvester B A Dehority MMorrison and J L Firkins ldquoTechnical note specific PCRamplification of protozoal 18S rDNA sequences from DNA

extracted from ruminai samples of cowsrdquo Journal of AnimalScience vol 81 no 3 pp 812ndash815 2003

[20] WHO Emerging Issues in Water and Infectious Disease WorldHealth Organization Geneva Switzerland 2003

[21] B Leiva E Clasdotter E Linder and J Winiecka-KrusnellldquoFree-living Acanthamoeba and Naegleria spp amebae in watersources of Leon Nicaraguardquo Revista de Biologıa Tropical vol56 no 2 pp 439ndash446 2008

[22] N Tsvetkova M Schild S Panaiotov et al ldquoThe identificationof free-living environmental isolates of amoebae fromBulgariardquoParasitology Research vol 92 no 5 pp 405ndash413 2004

[23] K A Coskun S Ozcelik L Tutar N Elaldi and Y TutarldquoIsolation and identification of free-living amoebae from tapwater in Sivas TurkeyrdquoBioMedResearch International vol 2013Article ID 675145 8 pages 2013

[24] S Onichandran T Kumar Y A L Lim et al ldquoWaterborneparasites and physico-chemical assessment of selected lakes inMalaysiardquo Parasitology Research vol 112 no 12 pp 4185ndash41912013

[25] M Adamska ldquoMolecular characterization of Cryptosporidiumand Giardia occurring in natural water bodies in PolandrdquoParasitology Research vol 114 no 2 pp 687ndash692 2015

[26] A P Balderrama-Carmona P Gortares-Moroyoqui L HAlvarez-Valencia et al ldquoQuantitative microbial risk assessmentof Cryptosporidium and Giardia in well water from a nativecommunity of Mexicordquo International Journal of EnvironmentalHealth Research pp 1ndash13 2014

[27] H Abd S Shanan A Saeed and G Sandstrom ldquoSurvival ofVibrio cholerae inside Acanthamoeba and detection of bothmicroorganisms from natural water samples may point outthe amoeba as a protozoal host for V choleraerdquo Journal ofBacteriology amp Parasitology vol S1-003 2012

[28] H Abd A Saeed A Weintraub and G Sandstrom ldquoVibriocholerae O139 requires neither capsule nor LPS O side chainto grow inside Acanthamoeba castellaniirdquo Journal of MedicalMicrobiology vol 58 no 1 pp 125ndash131 2009

[29] G Sandstrom A Saeed andH Abd ldquoAcanthamoeba polyphagais a possible host for Vibrio cholerae in aquatic environmentsrdquoExperimental Parasitology vol 126 no 1 pp 65ndash68 2010

[30] S Shanan H Abd I Hedenstrom A Saeed and G SandstromldquoDetection of Vibrio cholerae and Acanthamoeba species fromsame natural water samples collected from different choleraendemic areas in SudanrdquoBMCResearchNotes vol 4 article 1092011

[31] A L Newsome T M Scott R F Benson and B S FieldsldquoIsolation of an Amoeba naturally harboring a distinctiveLegionella speciesrdquo Applied and Environmental Microbiologyvol 64 no 5 pp 1688ndash1693 1998

[32] M Henke and K M Seidel ldquoAssociation between Legionellapneumophila and amoebae in waterrdquo Israel Journal of MedicalSciences vol 22 no 9 pp 690ndash695 1986

[33] S M Fletcher D Stark J Harkness and J Ellis ldquoEntericprotozoa in the developed world a public health perspectiverdquoClinical Microbiology Reviews vol 25 no 3 pp 420ndash449 2012

[34] P Muchesa O Mwamba T G Barnard and C Bartie ldquoDetec-tion of free-living amoebae using amoebal enrichment in awastewater treatment plant of gauteng province South AfricardquoBioMed Research International vol 2014 Article ID 575297 10pages 2014

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Signal TransductionJournal of


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Evolutionary BiologyInternational Journal of



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Advances in

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Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Number of positive samples by PCR utilizing primertargeting protozoal 18S rRNA gene

Location Positive samples Negative samples Total PrevalenceGadarif 19 114 133 143Juba 12 74 86 140Khartoum 6 60 66 91Kordofan 14 86 100 140Wad Madani 6 209 215 28Total 57 543 600 95

confirmed and a considerable number of waterborne out-breaks have been reported worldwide [12ndash14]

In Sudan Cryptosporidium species is an important causeof diarrhea in children and it is suggested that intrafamilialspread occurs [15] The highest prevalence of diarrhealdiseases was recorded among Port Sudan children (155)followed by children living in areas where people draw waterfrom unrectified hafirs (135) and finally children living inareas where water is drawn from rectified hafirs (60) [16]However there is no information about the presence of theprotozoal species except Cryptosporidium The aims of thisstudy were to detect protozoa from water samples by poly-merase chain reaction (PCR) using primer (P-SSU-342f andMedlin B) and identify species of the protozoa by sequencing

2 Material and Methods

21 SampleCollection 600 sampleswere collected fromwatersources (zeer hafir tank lake and stream) that belonged todifferent areas (Gadarif Khartoum Kordofan Juba andWadMadani) in Sudan for a one-year period in 2008 (Table 1Figure 1)

The hafir (an underground water reservoir designed forstoring rain water carried by streams and used for domesticwater supply and for agricultural purposes in rural areasin the Sudan) [17] is one of the most common sources ofwater in peripheral areas and different types of materials(contaminants) accumulate in it (due to erosion) as well asthe feces from wild and domestic animals The zeer is a tra-ditional Sudanese storage jug made of baked clay commonlyused to keep drinking water Water samples were collectedinto sterile 50mL tubes and transported to the laboratoryat Microbiology Department University of Medical Sciencesand Technology Khartoum

22 DNA Extraction The samples were centrifuged for10min at 4000 rpm The supernatant was removed and thesediment was used for DNA extraction The DNA wasextracted using routine proteinase K procedures using Qia-gen DNA mini kit (Qiagen Valencia CA USA)

23 DNA Amplification A protozoa-specific forward primer(P-SSU-342f) and Eukarya-specific reverse primer (MedlinB) targeting 18S rRNA gene were used The forward primerwas P-SSU-342f (51015840 CTTTCGATGGTAGTGTATTGGACT-AC-31015840) and the reverse primer was Medlin B (51015840-TGATCC-TTCTGCAGGTTCACCTAC-31015840) [18 19] in PCR to obtain

Figure 1 White color with arrows showing the surveyed areas inSudan and South Sudan

a product of about 1360 bp The PCR reaction performed at95∘C for 10min 35 cycles at 94∘C for 1min 50∘C for 1min72∘C for 1min final extension at 72∘C for 10min using 04120583Mof each primer The PCR amplification step was carried outand the final PCR reaction mixture was divided into twoparts One part was used for 2 agarose gel electrophoresiswith ethidium bromide staining and the other part was usedfor DNA sequencing

A castellanii was used as a positive control Detectionsensitivity was performed by serial dilution of A castellanii(10 cellsmL to 106 cellsmL)

24 Sequencing After samples collection DNA extractionand PCR analysis direct sequencing of the protozoa 18SrRNA gene was performed using purified nested PCR prod-ucts The sequencing was carried out by MWG operon(MWG operon Germany) The nucleotide sequences werethen edited and aligned using Blast program the sequenc-ing confirmed protozoal species according to the GenBankaccession numbers for their nucleotide sequences

25 Statistical Analysis 1205942 test was used to verify differencesin the existence of protozoa in water samples collected fromdifferent sources A 119875 value of le005 was considered statisti-cally significant

3 Results

The current study collected environmental water samplesand extracted amplified visualised and sequenced the PCRproducts using Blast program A detection limit of 10 cellswas found forAcanthamoeba according to the serial dilutions(Figure 2) The PCR amplification of the genomic DNAextracted from water samples produced a single band ofthe expected size of 1360 bp (Figure 3) Out of 600 watersamples 57 positive samples were detected by PCR and gel


M 101

102

103

104

105

106

1500bp


1 2 3 4 5 6 M

3000

1500

1000

500

400

300





4 Discussion














Gadarif


HafirC owczarzaki 1

C murisN caninum

11






13 0N caninum 1

A taiwanensis 1





21

O trifallaxS lemnae


E dispar 1


Total 19 17 25 5











References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M 101

102

103

104

105

106

1500bp


1 2 3 4 5 6 M

3000

1500

1000

500

400

300





4 Discussion














Gadarif


HafirC owczarzaki 1

C murisN caninum

11






13 0N caninum 1

A taiwanensis 1





21

O trifallaxS lemnae


E dispar 1


Total 19 17 25 5











References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Gadarif


HafirC owczarzaki 1

C murisN caninum

11






13 0N caninum 1

A taiwanensis 1





21

O trifallaxS lemnae


E dispar 1


Total 19 17 25 5











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 prevalence of protozoa species in

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