research article fasciola hepatica in some...

Research ArticleFasciola hepatica in Some Buffaloes andCattle by PCR and Microscopy

Sultan Ayaz1 Riaz Ullah2 Naser M AbdEl-Salam3 Sumiara Shams4 and Sadaf Niaz4

1 College of Veterinary Sciences and Animal Husbandry Abdul Wali Khan University Mardan Khyber Pakhtunkhwa 23200 Pakistan2Department of Chemistry Government College Ara Khel FR Kohat Khyber Pakhtunkhwa 26000 Pakistan3 Riyadh Community College King Saud University Riyadh 11437 Saudi Arabia4Department of Zoology Abdul Wali Khan University Mardan Khyber Pakhtunkhwa 23200 Pakistan

Correspondence should be addressed to Riaz Ullah afridiriazyahoocom

Received 30 June 2014 Revised 23 October 2014 Accepted 23 October 2014 Published 13 November 2014

Academic Editor Rafael Toledo

Copyright copy 2014 Sultan Ayaz 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

Fasciolosis is the burning problem of the livestock rearing community having huge morbidity mortality and economic losses tolivestock industries in our country Pakistan The faecal and liver biopsy samplings were examined by polymerase chain reaction(PCR) andmicroscopy technique during the entire study A total of 307 samples including 149 samples fromKarak and 158 samplesfromKohat abattoirs were examined by PCRmethod and overall prevalence of fasciolosis was 586 (18307) amongst theses 805(12149) in liver biopsy and 379 (6158) in feacal samples of cattle and Buffaloes were recorded Similarly the microscopy baseddetection was 358 (11307) including 461 (7149) in liver biopsy and 25 (4158) in faecal samples accordingly Furthermorethe areawise prevalence of fasciolosis in abattoirs by PCR method was found to be 759 (12158) in Kohat and 402 (6149) inKarak A 618 pb DNA was amplified in 2 agarose gel electrophoreses It is concluded from the study that prevalence of fasciolosiswas higher in abattoir of district Kohat and PCR was a more sensitive method of diagnosis than microscopy

1 Introduction

Fasciolosis in important food born and water born parasiticzoonosis caused by liver fluke of the genus Fasciola [1 2]the F hepatica is cosmopolitan in distribution with highfrequency in tropical area [3 4] Fasciola spp may reach thesize of 25ndash30mm in length and 8 to 15mm width It has leafshaped Structure [5] Fasciola hepatica has an interior andposterior sucker for attachment to host body [6] Fasciolahepatica completed its entire life cycle in two host cattlea definitive host and the snail an intermediate host whilethe human is an accidental host [1 7] which causes diseasemostly in ruminants especially in cattle buffaloes sheepgoats and cow It may however affect human [8]

These parasites inhabit the hepatobiliary system of theeffected animal and rarely can be found in ectopic sites withinthe host body [9] Once the parasites eggs are ingested by thecattle by the occasional drinking or grazing then the parasitesmigrate through the liver parenchyma to reach the bile ductThe diagnosis of fasciolosis in ruminant caused by Fasciola

spp has been made solely by the detection of Fasciola eggs inthe faeces of infected animal [10]

The worldwide losses in animal productivity due tofasciolosis were estimated as US $200 million per annumto rural agricultural communities and commercial produc-ers with over 600 million animals infected In developedcounties the incidence of F hepatica can reach up to 77In tropical countries fasciolosis is considered the singlemost important helminthes infection of cattle with reportedprevalence of 30ndash90 In domestic ruminants adverse effectsof acute or chronic fasciolosis include decreased meat andmilk production decreased fertility and increased veterinarycosts [11ndash13]

Fasciolosis is one of the big and most important world-wide problems mainly due to mortality of animals cost ofdiagnosis and treatment of condemned liver and it reducesmilk and meat production fertility disorder and drug resis-tance against fasciolosis [14]The present research project wasdesigned to carry out the PCR base prevalence of fasciolosisin cattle and buffaloes in abattoir of district Karak and Kohat

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 462084 5 pageshttpdxdoiorg1011552014462084

2 The Scientific World Journal

Table 1 Settings of PCR cycle for F hepatica

Stage Cycle Step Temperature Time1 1 1 92∘C 300min

2 251 92∘C 40 sec2 50∘C 40 sec3 72∘C 100min

3 1 1 92∘C 700min2 92∘C Hold

2 Materials and Methods

21 Samples Collection A total of 307 samples including 158faecal samples and 149 liver samples were collected from theabattoir of district Kohat and Karak Khyber Pakhtunkhwafrom the cattle having different sex and age Faeces sampleswere directly collected from the rectum of the cattle inpolythene bags which is duly labeled according to sex agedate and abattoir from which the samples were collected andsimilarly the liver samples were collected after slaughteringof those animal which are clinically suspected (having blisteror swelling on the liver surfaces) where the faecal samplesof the animals were collected The targeted swelling partsof the liver were incised with scalpel and put in a sterilizedbottle duly labeled with date spp sex and breed of theanimal The collected samples were placed in ice jar andwere immediately transported to the Virology andMolecularParasitology Laboratory of the Zoology Department KohatUniversity of Science and Technology Kohat

22 Microscopy Thick and thin smears were prepared fromthe faecal and liver biopsy including bile duct material Bothof faeces and liver biopsy were mixed with buffer saline and adrop of 20120583Lwas placed on the slides anddried andwere putsa drop of immersion oil and then observed under the directmicroscopy of 10x 40x and 100xThe images were comparedwith the standard morphological size

23 DNA Extraction The samples were subjected to DNAextraction by using GF-1 kit (vivantis) as per the manufac-turer protocol (Sultan Ayaz PhD thesis 2009 HEC Pakistanpanel) A 200 120583L liver biopsy as well as the faecal pellet sam-ples in Eppendorf tube was mixed with 50120583L of proteinaseK and 200 120583L of buffer VL They were mixed well with thehelp of vertex and then were incubated at 65∘C for 10minin hot plates The columns were centrifuged at 6000 rpm for1min and the flow through was discarded then 200120583L ofwash buffer was added and centrifuged at 6000 rpm for 1minand again the supernatant was discarded Similarly 200120583Lof wash buffer 2 was added and centrifuged at 6000 rpmfor 1min and supernatant was discarded Then the columnswere transferred to new tubes and 30 120583L of elution bufferwas added and placed for 2min at room temperature Afterthat centrifugation was at 6000 rpm for 1min and was mixedwith 30 120583L of deionized water and stored at minus80∘C for furtherprocess

050

100150200250300350

ss kk kt

LiverFaecal

Total

Figure 1 Prevalence of fasciolosis cattle and buffaloes by using PCRand microscopy methods in Karak and Kohat Pakistan

24 DNA Amplification (PCR) The DNA was amplifiedthrough polymerase chain reaction (PCR) using primerspecific for Fasciola hepatica (Figure 2) The primer addedfor F hepatica was -F 51015840-AGTGATTACCCGCTGAACT-31015840 and R 31015840-CTGAGAAAGTGCACTGACAA-51015840 [13] Thespecific amplified product was compared with 100 bp DNAladder marker (Fermentas USA) The parasitic DNA wasrecognized

The target DNA was amplified in 20120583L reaction mix-ture containing 10x PCR buffer 2 120583M 1 120583M deoxynucleosidetriphosphate (500120583M) 24 120583MMgCl

2(25 120583m) 1 120583M primers

(10 pmol) target DNA 5 120583L and 03 unit of Taq DNA poly-merase (5 u120583L) add deionizedwater up to 20 120583LDenaturingof DNA amplification was done at temperature (92∘C for3min 25 cycles) (92∘C for 40 sec) (50∘C for 40 sec) and(72∘C for 60 sec) In the last stage extension at 72∘C for 7minand hold at 4∘C for unlimited time (Table 1) the designedprogram was saved

25 Gel Electrophoresis In gel electrophoresis 2 g of Agarosewas added in 100mL of TBE buffer and placed in oven for 2minutes at 100∘CThen removed this mixture and cool downit up to 45∘C after then added 20120583L of ethidium bromideThe gel was poured into gel tray and combs were fixedCombs were removed after gel was formed So by this waythe Gel tray was placed in gel tank containing 1000mL 05xTBE buffer 10120583L Of PCR product was mixed with 5 120583L ofbromophenol blue dye of each sample was loaded in the wellsand 15 120583L of DNA ladder (100 bp) was loaded in the separatewell The positive and negative control was run parallel withthe samples The gel was run for 25min at voltage of 130volts and 500 ampere current Gel was then examined by UVtransilluminator A photo was cached and saved in a record

3 Results and Discussion

Fasciolosis is a very serious disease having huge economiclosses of the cattle and industries in terms of meat milk andleather in our country In the current study a total of 307samples were examined which included 149 samples fromKarak and 158 samples from Kohat of the cattle and buffalosof Khyber Pakhtunkhwa By examination it is shown that theoverall prevalence of fasciolosis was 586 (18307) amongst

The Scientific World Journal 3

Table 2 Prevalence of fasciolosis in cattle and buffaloes in district abattoirs of Karak and Kohat by using PCR and microscopy methods

SampleSpp

cattle + buffaloesfrom Karak + Kohat

KarakPCR positive

(cattle + buffaloes)

KohatPCR positive


Prevalence(PCR)

Microscopyprevalence

Liver sample 70 + 79 = 149 4571 (470)

81012 (879)

4 + 8 = 12805 (12149)

714946

Faecal sample 79 + 79 = 158 2253 (279)

4506 (479)

2 + 4 = 6379 (6158)

415825

G total 149 + 148 = 307 4 + 2 = 6402 (6149)

8 + 4 = 12759 (12158)

6 + 12 = 18586 (18307)

11307358

Table 3 PCR based detection of fasciolosis in the district abattoir of Kohat

SampleCow Buffalo Total sample Prevalence Total

prevalence Other findingsPositivesample

Negativesample

Positivesample

Negativesample Cow Buffaloes Cow Buffalo

Faecalsample 1 38 3 37 39 40 256

(139)75(340) 506 (479)

E histolyticaand

G lambliacryptosporidium

Liversample 3 35 5 36 38 41 833

(338)1219(541) 1012 (879) No other findings

G total 4 73 8 76 77 81 519(477)

98(881) 759 (12158)

E histolyticaand


Table 4 PCR based detection of fasciolosis in the district abattoir of Karak



Negativesample

Positivesample


Faecalsample 1 39 1 38 40 39 25

(140)256(139) 253 (279)

E histolyticaand


Liversample 1 24 3 42 25 45 4


G total 2 63 4 80 65 84 307(265)

476(484) 402 (6149)

E histolyticaand


1 2 3 4 5 6 7 8 9 10 11 12 13 14 M

618 bp

Figure 2 M 100 bp DNA ladder lane 5 is positive control while lane 14 is negative control Lanes 13 12 11 9 and 8 are positive and the otherlanes 1 2 3 4 6 7 and 10 are negative The amplified DNA band showing 618 bp


these 402 (6149) in the district Karak and 75 (12158) inthe district Kohat Furthermore the prevalence of Fasciolosisin cows was 307 (265) and 476 (484) in the Buffaloesof the district Karak was recorded while 519 (477) in theCow and 987 (881) in the Buffaloes of district Kohat ofthe Khyber Pakhtunkhwa were recorded The prevalence offasciolosis was higher in district Kohat as compared to districtKarak Statistical analysis revealed the significant difference119875 lt 005 when the data was interpreted (Tables 2 3 and 4)(Figure 1)

In the present study F hepatica was found in the fecalsample and liver biopsy sample of cows and buffaloes inthe abattoir of the district Karak and Kohat of the KhyberPakhtunkhwa province of Pakistan

One of the studies revealed that it was the diseaseof domesticated animals in Sindh province that causesheavy infection of F hepatica Moreover F gigantica wasreported at high altitudes in Khyber Pakhtunkhwa provincewhereas F hepatica occurred in deltaic region of Punjaband Sindh provinces Pakistan Similar findings were pre-viously reported In Faisalabad (central Punjab) [15] overallprevalence of fasciolosis was found to be 1755 of which Fhepatica was 57 However mixed infection was revealed in202 animals [16]

Fasciola hepatica was the dominant fluke species in theanimals [17] This may be associated with the existence offavorable ecological biotopes for Lymnaea truncatula therecognized intermediate host of F hepatica in Ethiopia [18]

The worldwide losses in animal productivity due to fasci-olosis were estimated at US $200 million per annum to ruralagricultural communities and commercial producers [19]with over 600 million animals infected [20] In developedcounties the incidence of F hepatica can reach up to 77In tropical countries fasciolosis is considered the singlemost important helminthes infection of cattle with reportedprevalence of 30ndash90 [21] This study coincided partiallywith our study in cattle and buffaloes

Similar study from Northern Iran (Tonekabon) indicatesa 15 prevalence of fasciolosis in buffaloes and 46 incattle [22] Fasciolosis is now recognized as an emerging foodborn zoonosis in many parts of the world and world healthorganization has also included human fasciolosis on its list[23]

Conflict of Interests

The authors declared that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors are thankful to the Deanship of ScientificResearch King SaudUniversity Riyadh for funding the workthrough the research group Project no RGP-210

References

[1] S Mas-Coma M D Bargues and M A Valero ldquoFascioliasisand other plant-borne trematode zoonosesrdquo International Jour-nal for Parasitology vol 35 no 11-12 pp 1255ndash1278 2005

[2] P ZhouNChen R-L Zhang R-Q Lin andX-Q Zhu ldquoFood-borne parasitic zoonoses in China perspective for controlrdquoTrends in Parasitology vol 24 no 4 pp 190ndash196 2008

[3] M S Mas-Coma J G Esteban and M D Bargues ldquoEpi-demiology of human fascioliasis a review and proposed newclassificationrdquo Bulletin of theWorld Health Organization vol 77no 4 pp 340ndash346 1999

[4] T W Spithill and J P Dalton ldquoProgress in development of liverfluke vaccinesrdquo Parasitology Today vol 14 no 6 pp 224ndash2281998

[5] S J AndrewsThe Life Cycles of Fasciola hepatica edited by J PDalton CAB International Oxon UK 1999

[6] A M Dunn Veterinary Helminthology Butler and TannerLondon UK 2nd edition 1978

[7] J L Vaughan J A Charles and J C Boray ldquoFasciola hepaticainfection in farmed emus (Dromaius novaehollandiae)rdquo Aus-tralian Veterinary Journal vol 75 no 11 pp 811ndash813 1997

[8] H Alcaino ldquoEpidemiology of fasciolasis in chilerdquo in BasicResearch in Helminthiasis R Ehrlich A Nieto and L YarzabalEds pp 11ndash30 Ediciones Logos Montevideo Uruguay 1990

[9] T G T Nguyen N van de J Vercruysse P Dorny and T HLe ldquoGenotypic characterization and species identification ofFasciola spp with implications regarding the isolates infectinggoats in Vietnamrdquo Experimental Parasitology vol 123 no 4 pp354ndash361 2009

[10] S J Andrews ldquoThe life cycle of Fasciola hepaticardquo in FasciolosisJ P Dalton Ed pp 1-2 CABI Publishing Oxin UK 1999

[11] G Theodoropoulos E Theodoropoulou G Petrakos V Kant-zoura and J Kostopoulos ldquoAbattoir condemnation due toparasitic infections and its economic implications in the regionof Trikala Greecerdquo Journal of Veterinary Medicine vol 49 no6 pp 281ndash284 2002

[12] E S Swai and E Ulicky ldquoAn evaluation of the economiclosses resulting from condemnation of cattle livers and loss ofcarcass weight due to Fasciolosis a case study from Hai townabattoir Kilimanjaro region Tanzaniardquo Livestock Research forRural Development vol 21 no 11 2009

[13] I Khan AMKhan S Ayaz S KhanMAnees and S A KhanldquoMolecular detection of Fasciola hepatica in water sources ofDistrict Nowshehra Khyber Pakhtunkhwa Pakistanrdquo Interna-tional Journal of Advancements in Research amp Technology vol1 no 7 pp 1ndash11 2012

[14] J Keiser and J Utzinger ldquoFood-borne trematodiasis currentchemotherapy and advances with artemisinins and synthetictrioxolanesrdquo Trends in Parasitology vol 23 no 11 pp 555ndash5622007

[15] S B Kendall ldquoFasciolosis in Pakistanrdquo Annals of TropicalMedicine and Parasitology vol 48 pp 307ndash313 1954

[16] C S Hayat Z Iqbal B Hayat and M N Khan ldquoStudies onthe seasonal prevalence of fasciolosis and lungworm disease insheep at Faisalabadrdquo Pakistan Vaternary Journal vol 6 no 3 p131 1986

[17] M Graber and P Daynes ldquoMollusques vecteurs de trema-todoses humaines et animals en Ethiopierdquo Revue drsquoElevage etde Medecine Veterinaire des Pays Tropicaux vol 27 no 3 pp307ndash322 1974

[18] J C Boray ldquoFlukes of domestic animalsrdquo in Parasites Pests andPredators S M Gaafar W S Howard and R E Marsh Edspp 178ndash218 Elsevier New York NY USA 1985


[19] V Ramajo A Oleaga P Casanueva G V Hillyer and A MuroldquoVaccination of sheep against Fasciola hepatica with homolo-gous fatty acid binding proteinsrdquo Veterinary Parasitology vol97 no 1 pp 35ndash46 2001

[20] T W Spithill P M Smooker and D B Copeman ldquoFasciolagigantica epidemiology control immunology and molecularbiologyrdquo in Fasciolosis J P Dalton Ed 1999

[21] A M Phiri I K Phiri C S Sikasunge and J MonradldquoPrevalence of fasciolosis in Zambian cattle observed at selectedabattoirs with emphasis on age sex and originrdquo Journal ofVeterinary Medicine Series B vol 52 no 9 pp 414ndash416 2005

[22] A Freites C Colmenares B Alarcon-Noya M E Garcıa andO Dıaz-Suarez ldquoHuman fasciolosis inMaramunicipality Zuliastate Venezuela prevalence and asociated factorsrdquo InvestigacionClinica vol 50 no 4 pp 497ndash506 2009

[23] World Health Organization Fact Sheet on Fasciolosis ActionAgainst Worms vol 10 World Health Organization Headquar-ters Geneva Switzerland 2008

Submit your manuscripts athttpwwwhindawicom

Veterinary MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International



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Evolutionary BiologyInternational Journal of


Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

Biotechnology Research International


Agronomy




Journal of Parasitology Research

Hindawi Publishing Corporation httpwwwhindawicom


Volume 2014

Zoology

GenomicsInternational Journal of


InsectsJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


VirusesJournal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of



Case Reports in Veterinary Medicine


Table 1 Settings of PCR cycle for F hepatica

Stage Cycle Step Temperature Time1 1 1 92∘C 300min

2 251 92∘C 40 sec2 50∘C 40 sec3 72∘C 100min

3 1 1 92∘C 700min2 92∘C Hold

2 Materials and Methods

21 Samples Collection A total of 307 samples including 158faecal samples and 149 liver samples were collected from theabattoir of district Kohat and Karak Khyber Pakhtunkhwafrom the cattle having different sex and age Faeces sampleswere directly collected from the rectum of the cattle inpolythene bags which is duly labeled according to sex agedate and abattoir from which the samples were collected andsimilarly the liver samples were collected after slaughteringof those animal which are clinically suspected (having blisteror swelling on the liver surfaces) where the faecal samplesof the animals were collected The targeted swelling partsof the liver were incised with scalpel and put in a sterilizedbottle duly labeled with date spp sex and breed of theanimal The collected samples were placed in ice jar andwere immediately transported to the Virology andMolecularParasitology Laboratory of the Zoology Department KohatUniversity of Science and Technology Kohat

22 Microscopy Thick and thin smears were prepared fromthe faecal and liver biopsy including bile duct material Bothof faeces and liver biopsy were mixed with buffer saline and adrop of 20120583Lwas placed on the slides anddried andwere putsa drop of immersion oil and then observed under the directmicroscopy of 10x 40x and 100xThe images were comparedwith the standard morphological size

23 DNA Extraction The samples were subjected to DNAextraction by using GF-1 kit (vivantis) as per the manufac-turer protocol (Sultan Ayaz PhD thesis 2009 HEC Pakistanpanel) A 200 120583L liver biopsy as well as the faecal pellet sam-ples in Eppendorf tube was mixed with 50120583L of proteinaseK and 200 120583L of buffer VL They were mixed well with thehelp of vertex and then were incubated at 65∘C for 10minin hot plates The columns were centrifuged at 6000 rpm for1min and the flow through was discarded then 200120583L ofwash buffer was added and centrifuged at 6000 rpm for 1minand again the supernatant was discarded Similarly 200120583Lof wash buffer 2 was added and centrifuged at 6000 rpmfor 1min and supernatant was discarded Then the columnswere transferred to new tubes and 30 120583L of elution bufferwas added and placed for 2min at room temperature Afterthat centrifugation was at 6000 rpm for 1min and was mixedwith 30 120583L of deionized water and stored at minus80∘C for furtherprocess

050

100150200250300350

ss kk kt

LiverFaecal

Total

Figure 1 Prevalence of fasciolosis cattle and buffaloes by using PCRand microscopy methods in Karak and Kohat Pakistan

24 DNA Amplification (PCR) The DNA was amplifiedthrough polymerase chain reaction (PCR) using primerspecific for Fasciola hepatica (Figure 2) The primer addedfor F hepatica was -F 51015840-AGTGATTACCCGCTGAACT-31015840 and R 31015840-CTGAGAAAGTGCACTGACAA-51015840 [13] Thespecific amplified product was compared with 100 bp DNAladder marker (Fermentas USA) The parasitic DNA wasrecognized

The target DNA was amplified in 20120583L reaction mix-ture containing 10x PCR buffer 2 120583M 1 120583M deoxynucleosidetriphosphate (500120583M) 24 120583MMgCl

2(25 120583m) 1 120583M primers

(10 pmol) target DNA 5 120583L and 03 unit of Taq DNA poly-merase (5 u120583L) add deionizedwater up to 20 120583LDenaturingof DNA amplification was done at temperature (92∘C for3min 25 cycles) (92∘C for 40 sec) (50∘C for 40 sec) and(72∘C for 60 sec) In the last stage extension at 72∘C for 7minand hold at 4∘C for unlimited time (Table 1) the designedprogram was saved

25 Gel Electrophoresis In gel electrophoresis 2 g of Agarosewas added in 100mL of TBE buffer and placed in oven for 2minutes at 100∘CThen removed this mixture and cool downit up to 45∘C after then added 20120583L of ethidium bromideThe gel was poured into gel tray and combs were fixedCombs were removed after gel was formed So by this waythe Gel tray was placed in gel tank containing 1000mL 05xTBE buffer 10120583L Of PCR product was mixed with 5 120583L ofbromophenol blue dye of each sample was loaded in the wellsand 15 120583L of DNA ladder (100 bp) was loaded in the separatewell The positive and negative control was run parallel withthe samples The gel was run for 25min at voltage of 130volts and 500 ampere current Gel was then examined by UVtransilluminator A photo was cached and saved in a record

3 Results and Discussion

Fasciolosis is a very serious disease having huge economiclosses of the cattle and industries in terms of meat milk andleather in our country In the current study a total of 307samples were examined which included 149 samples fromKarak and 158 samples from Kohat of the cattle and buffalosof Khyber Pakhtunkhwa By examination it is shown that theoverall prevalence of fasciolosis was 586 (18307) amongst



SampleSpp


KarakPCR positive


KohatPCR positive


Prevalence(PCR)


Liver sample 70 + 79 = 149 4571 (470)

81012 (879)

4 + 8 = 12805 (12149)

714946

Faecal sample 79 + 79 = 158 2253 (279)

4506 (479)

2 + 4 = 6379 (6158)

415825

G total 149 + 148 = 307 4 + 2 = 6402 (6149)

8 + 4 = 12759 (12158)

6 + 12 = 18586 (18307)

11307358




Negativesample

Positivesample


Faecalsample 1 38 3 37 39 40 256

(139)75(340) 506 (479)

E histolyticaand


Liversample 3 35 5 36 38 41 833


G total 4 73 8 76 77 81 519(477)

98(881) 759 (12158)

E histolyticaand





Negativesample

Positivesample


Faecalsample 1 39 1 38 40 39 25

(140)256(139) 253 (279)

E histolyticaand


Liversample 1 24 3 42 25 45 4


G total 2 63 4 80 65 84 307(265)

476(484) 402 (6149)

E histolyticaand


1 2 3 4 5 6 7 8 9 10 11 12 13 14 M

618 bp











Acknowledgment


References
































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of








SampleSpp


KarakPCR positive


KohatPCR positive


Prevalence(PCR)


Liver sample 70 + 79 = 149 4571 (470)

81012 (879)

4 + 8 = 12805 (12149)

714946

Faecal sample 79 + 79 = 158 2253 (279)

4506 (479)

2 + 4 = 6379 (6158)

415825

G total 149 + 148 = 307 4 + 2 = 6402 (6149)

8 + 4 = 12759 (12158)

6 + 12 = 18586 (18307)

11307358




Negativesample

Positivesample


Faecalsample 1 38 3 37 39 40 256

(139)75(340) 506 (479)

E histolyticaand


Liversample 3 35 5 36 38 41 833


G total 4 73 8 76 77 81 519(477)

98(881) 759 (12158)

E histolyticaand





Negativesample

Positivesample


Faecalsample 1 39 1 38 40 39 25

(140)256(139) 253 (279)

E histolyticaand


Liversample 1 24 3 42 25 45 4


G total 2 63 4 80 65 84 307(265)

476(484) 402 (6149)

E histolyticaand


1 2 3 4 5 6 7 8 9 10 11 12 13 14 M

618 bp











Acknowledgment


References
































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of















Acknowledgment


References
































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of



















Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of






research article fasciola hepatica in some...

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