Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 868050 6 pageshttpdxdoiorg1011552013868050

Research ArticleMolecular Identification of Ancylostoma caninum Isolated fromCats in Southern China Based on Complete ITS Sequence

Yuanjia Liu1 Guochao Zheng1 Muhamd Alsarakibi1 Xinheng Zhang2 Wei Hu1

Pengyun Lu1 Liqin Lin1 Liping Tan1 Qin Luo1 and Guoqing Li1

1 College of Veterinary Medicine South China Agricultural University Guangzhou 510642 China2 College of Animal Science South China Agricultural University Guangzhou 510642 China

Correspondence should be addressed to Guoqing Li gqliscaueducn

Received 11 April 2013 Revised 7 July 2013 Accepted 22 August 2013

Academic Editor Markus Schuelke

Copyright copy 2013 Yuanjia Liu 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

Ancylostoma caninum is a blood-feeding parasitic intestinal nematode which infects dogs cats and other mammals throughoutthe world A highly sensitive and species-specific PCR-RFLP technique was utilised to detect the prevalence of A caninum in catsin Guangzhou southern China Of the 102 fecal samples examined the prevalence of A caninum in cats was 951 and 833using PCR-RFLP and microscopy respectively Among them the prevalence of single hookworm infection with A caninum was5490 while mixed infections with both A caninum and A ceylanicumwere 4020 Comparative analysis of three complete ITSsequences obtained from cat-derivedA caninum showed the same length (738 bp) as that of dog-derivedA caninum However thesequence variation range was 986ndash100 where only one cat isolate (M63) showed 100 sequence similarity in comparison withtwo dog-derived A caninum isolates (AM850106 EU159416) in the same studied area The phylogenetic tree revealed A caninumderived from both cats and dogs in single cluster Results suggest that cats could be the main host of A caninum in China whichmay cause cross-infection between dogs and cats in the same area

1 Introduction

Ancylostoma caninum is a blood-feeding parasitic intestinalnematode which infects dogs cats and other mammalsthroughout the temperate and tropical areas in the world [1ndash3] In addition to the veterinary importance A caninum canalso cause zoonotic disease in humans The larvae of Acaninum hatch from eggs and develop into infective larvae viatwo molts The infective larvae then infect host animals suchas dogs and cats migrate into the intestine and develop intoadult worms following two more molts If the infective larvaeinvade humans they can cause cutaneous larvae migrans(CLM) or ldquocreeping eruptionsrdquo which are hypersensitivereactions in response to the migration of A caninum larvaehowever they cannot develop into adult worms just bymigrating under the skin [4]

Although some cases recorded thatA caninumwas foundin cats [5ndash8] this species has been still regarded as an uncom-mon parasite of cats Therefore A caninum was describedas ldquodog hookwormrdquo [9] and was supposed as a host-specific

parasite for canids [2 10] while Palmer stated that Acaninum was the predominant species of hookworm in dogs[11] and A tubaeforme was the predominant species ofhookworm in cats In China high occurrence of A caninumhas been reportedwith prevalence of 104ndash73 but withoutsignificant area differences [12 13] Although A caninum incats has been reported inThailand (23) [14] Australia (30)[11] and Sichuan province in China (25 and 51) [15 16]data on prevalence of A caninum in cats are still scarce

Herein this study presents the first molecular identifica-tion based on complete ITS sequence as well as it describesa simple and effective detection method for A caninum fromcats in southern China

2 Materials and Methods

21 Area Studied Guangzhou city is located in south-centralGuangdong province (N 22∘451015840 sim23∘051015840 E 113∘141015840 sim113∘341015840)southernChina experiencing a typically tropical climatewith

2 BioMed Research International

Table 1 Predictive restriction patterns by endonucleases EcoR II BsuR I and Taq I at ITS1 and 58S locus

Species PCR amplicon (bp) Cleavage site Predicted fragment size (bp)EcoR II BsuR I Taq I

A caninum 404 minus minus 3+ 60307 (12U 25U)A tubaeforme 405 minus minus 2+ 60333 (12U)A ceylanicum 404 + minus 76328A braziliense 408 2+ minus 76122210U stenocephala 406 minus + 87319UMeans that the fragment is too small to visible

heavy monsoon rains It covers an area of approximately8000 km2 divided into 10 geographical districts with anestimated population of approximately 12 million This citycontains a large number of sheltered cats and owns twolocal humane shelters for stray cats in Conghua and Baiyundistricts

22 Fecal Sample Collection and Processing Cat fecal samples(119899 = 102) were collected from Conghua (119899 = 72) and Baiyun(119899 = 30) humane shelters during March and July 2012 Asingle sample was collected in clean container from eachcat directly transported to laboratory preserved in 25potassium dichromate and stored at 4∘C To detect thepresence of hookworm eggs direct microscopic examinationwas done by saturated sodium chloride and glucose flotationPositive fecal samples were further characterized by molecu-lar procedures

23 Genomic DNA Extraction DNAs were extracted directlyfrom fecal samples using a commercial DNA extraction kit(QIAamp DNA Stool Mini Kit QIAgen Hilden Germany)according to the manufacturerrsquos protocols However allsamples were pretreated with 5 cycles of heating at 100∘Cfor 5 minutes followed by immediate freezing at minus80∘Cfor 5 minutes A negative control (water) was used in eachextraction group Extracted DNAs were then stored at minus20∘C

24 Primers and Restriction Enzyme One pair of primersAF (51015840-CTTTGTCGGGAAGGTTGG-31015840) and AR (51015840-TTC-ACCACTCTAAGCGTCT-31015840) was designed from conservedregion of ITS sequences of five species of hookwormsincluding A caninum (AM85010 AM850105) A tubae-forme (JQ812691) A ceylanicum (DQ381541 DQ780009)A braziliense (DQ359149 DQ438056) and U stenocephala(HQ262053 AF194145) by Primer Premier 50 to amplify404 bp region ofA caninum 405 bp ofA tubaeforme 408 bpof A braziliense 404 bp of A ceylanicum and 406 bp of Ustenocephalawhich contain ITS1 and 58S rRNA regionsThefive species of hookworms could be distinguished by restric-tion endonucleases EcoRII BsuRI and Taq I for differentcutting sites on the sequence according to the analysis byPrimer Premier 50 The theoretical cutting patterns of thefive different hookworm DNA fragments treated by threerestriction enzymes are shown in Table 1 EcoR II couldidentify A ceylanicum and A braziliense while BsuRI canonly identify U stenocephala and Taq I was distinguishedbetween A caninum and A tubaeforme

Another pair of primers CAF (51015840-GACTGCGGACTG-CTGTAT-31015840) and CAR (51015840-AAGTTCAGCGGGTAGTCA-31015840) was designed by Primer Premier 50 based on ITSsequences (JQ812694 AJ920347 and AM039739) of A can-inum to amplify the complete ITS sequence of cat-derived Acaninum

25 PCR-RFLP Both PCRs were performed in 25 120583L volumecontaining 2 120583L of the DNA sample 02120583L ofTaq polymerase(TaKaRa Dalian China) 25 120583L of 10timesTaq buffer (TaKaRa)2 120583L of diethylnitrophenyl thiophosphate (dNTP TaKaRa)mixture 05 120583L of each primer (AFAR or CAFCAR50mM) and 173 120583L of distilled water PCR cycling param-eters were as follows 1 cycle at 96∘C for 5 minutes then 35cycles of 96∘C for 30 seconds at 60∘C for 30 seconds and at72∘C for 90 seconds followed by 1 cycle at 72∘C for 7minutes

RFLP analysis was performed by digesting 7 120583L of PCRproduct with 2U of each restriction endonuclease (TaKaRa)in a final volume of 20120583L for 3 hours at 37∘C PCR productsand restriction fragments were analyzed after electrophoresisin 2 and 3 agarose gels with 02 120583gmL of ethidium bro-mide staining and were visualized on a UV transilluminator

26 Sequence Confirmation and Phylogenetic Analysis Posi-tive amplicons were purified and sequenced using ABI 3730automated DNA sequencer (BigDye Terminator Chemistry)Obtained sequences were aligned with 15 ITS referencesequences using Clustal X programs [17] Phylogenetic treeswere constructed usingMEGA version 51 (MEGA51Molec-ular Evolutionary Genetics Analysis software Arizona StateUniversity Tempe Arizona USA) Bootstrap analyses wereconducted using 1000 replicates to assess the reliability ofinferred tree topologies Neighbor-joining algorithms wereconducted using the Kimura 2 parameter distance analysisObtained nucleotide sequences have been deposited in theGenBank database under accession numbers KC755015 andKC755025

3 Results

Of the 102 collected fecal samples 85 samples (833) weremicroscopically positive while 97 samples (951) were PCRpositive for hookwormThe prevalence of A caninum in catsfrom suburban area (861 Conghua) was higher than thatfrom urban area (767 Baiyun)

The results showed that U stenocephala was absent in the97 examined PCR samples where there was no enzymaticdigestion by restriction endonucleases BsuRI (not shown)

BioMed Research International 3

Figure 1 PCR amplicons digested by restriction endonucleases EcoR II Lanes 1ndash62 samples from Conghua district lanes 62ndash85 samplesfrom Baiyun district lanes 86ndash97 microscopically negative samples M DL-2000 DNA marker

Figure 2 PCR amplicons digested by restriction endonucleases Taq I Lanes 1ndash53 random samples from 56 samples P PCR amplicon MDL-2000 DNA marker

Cat-China-KC755025Cat-China-KC755022Dog-China-A caninum-AM850106Dog-China-A caninum-EU159416Cat-China-KC755018

50 Dog-China-A caninum-EU159415Dog-China-A caninum-AM850105

Cat-China-KC755023Dog-America-A caninum-JQ812694

60 Cat-China-KC755019Cat-China-KC755024

42 Cat-China-KC75501751 35 Cat-China-KC755016

Human-China-A duodenale-EU344797Cat-America-A tubaeforme-JQ812691

Dog-America-A braziliense-JQ81269259 Cat-America-A braziliense-JQ812693

Cat-China-KC755020Dog-India-A ceylanicum-DQ780009

77 Dog-America-A ceylanicum-DQ381541Cat-China-KC755021Cat-China-KC755015

Urocyon-USA-U stenocephala-HQ262053Urocyon-USA-U stenocephala-HQ262054

92

64

9694

35

87

100

663370

100

0005

A braziliense

U stenocephala

A caninum

A ceylanicum

Figure 3 Phylogenetic tree of hookworm isolates based on the ITS1 and 58S rRNA sequences with neighbor-joining algorithm using Kimuratwo-parameter The reference sequences are available in the GenBank by their accession numbers The isolates of this study are shown in redcolor

Figure 1 shows the digestion results of the 97 PCR ampliconsby restriction endonucleases EcoR II in which 41 samplesshowed three bands revealing that these samples weremixed infections with A ceylanicum and one undeterminedhookworm (A caninum or other) while 56 samples showedone band revealing that these samples were single infectionwith one undetermined hookworm (A caninum or other)Fifty three samples from the 56 single infection samples wererandomly chosen for the Taq I digestion the results showthat all these samples were infected with only one species(Figure 2)Thereafter 11 positive samples (656 and 541)weresuccessfully sequenced the phylogenetic tree based on those

sequences with 15 reference sequences (Figure 3) revealedthat the 56 positive samples were infected with A caninumwhile the 41 samples were infected with both A ceylanicumand A caninum without any A tubaeforme infection in ourstudy Thus the overall prevalence of A caninum infectionwas 951 with a prevalence of 549 single infection and402 mixed infections with A ceylanicum

Three complete ITS sequences from cat-derived A can-inum isolates (M45 M63 and M84) were obtained byPrimer CAFCAR and submitted in the GenBank underaccession numbers (KC755026 KC755028 and KC755029)All ITS sequences were 738 bp in length which was the same

4 BioMed Research International

Table 2 Previous investigations of hookworm species in cats worldwideCountry (area) Detection method Species Prevalence Source of animal Reference

Australia (Sydney) Necropsy A tubaeforme 35 Pound [6]U stenocephala 05 (119899 = 404)

Australia (Tasmania) Necropsy U stenocephala 2 (119899 = 86) Feral cats [18]Australia (Brsibane) Necropsy A tubaeforme 19 (119899 = 404) Pound [7]Australia (Brisbane) Necropsy A tubaeforme 81 Pound [19]Australia (Adelaide) Fecal exam U stenocephala 03 (119899 = 376) Vet practice [20]Australia (Kimberley) Necropsy A tubaeforme 20 (119899 = 34) Aboriginal community [21]Australia (Northern territory) Necropsy A tubaeforme 13 Feral cats [22]

Australia (countrywide) PCR-RFLP A tubaeforme 70 (119899 = 10) Refuge and pet cats [11]A caninum 30 (119899 = 10)

Brazil (Rio de Janeiro) Necropsy A braziliense 659 (119899 = 135) Cat (shelters and ZoonosesControl Center)

[23]A tubaeforme 89 (119899 = 135)

China (Sichuan) Necropsy

A caninum 25 (119899 = 20)

Domestic cat [15]A braziliense 10 (119899 = 20)A ceylanicum 5 (119899 = 20)U stenocephala 10 (119899 = 20)

China (Sichuan) NecropsyA caninum 51 (119899 = 149)

Domestic cat [16]A braziliense 174 (119899 = 149)U stenocephala 14 (119899 = 149)

Costa Rica (San Isidro ELGeneral) Fecal exam A tubaeforme 11 (119899 = 9) Refuge cat [24]

Thailand (Prachin Buri) A ceylanicumA caninum

9223 Cat [14]

Italy (central) Larvae (L3) exam A tubaeforme 12 (119899 = 81) Cat (pet) [25]U stenocephala 37 (119899 = 81)

Malaysia (West Malaysia) PCR-sequencing A ceylanicumA braziliense

261 (119899 = 23)43 (119899 = 23) Domestic cat [26]

Qatar (Doha) Necropsy A tubaeforme 147 (119899 = 658) Cat (feral) [27]Spain (mid-Ebro Valley) Necropsy A tubaeforme 293 (119899 = 58) Cat (stray) [28]

USA (Florida) Necropsy A tubaeforme 75 (119899 = 60) Cat (feral) [29]A braziliense 33 (119899 = 60)

USA (Pennsylvania) Fecal exam A tubaeforme 05 (119899 = 1566) Cat (pet) [30]

length presented by the A caninum ITS region (AM850105AM850106 EU159415 and EU159416) isolated from dogs inthis area DNA sequences were assembled using DNAStar(version 7 Madison WI USA) and multiple-sequence align-ment was performed with MegAlign program Comparativeanalysis of the three complete ITS sequences obtained fromcat-derived A caninum showed the same length (738 bp) ofdog-derived A caninum with a similarity of 992ndash997However compared to the ITS sequences of dog-derivedA caninum isolates (AM850105 AM850106 EU159415 andEU159416) the sequence variation range was 986ndash100where only one cat isolate (M63) showed 100 sequencesimilarity compared with two dog-derived A caninum iso-lates (AM850106 and EU159416) in the same studied areaThe sequence similarities of the cat-derived A caninum withA ceylanicum (KC755027) A tubaeforme (JQ812691) and Abraziliense (JQ812692) were 970ndash973 973ndash976 and916ndash921 respectively

4 Discussion

As early as the 19th century hookworms in cats and dogshad been described by Zedler (1800) and Ercolani (1859)[31] For nearly 100 years the common hookworm of bothdogs and cats was referred as Ancylostoma caninum [32]Later then some authors stated that it was difficult to infectdogs with larvae from cats and vice versa [32ndash36] Studiesin Europe Africa and Australia described themorphologicaldifferences of hookworms obtained from dogs and catswhere A caninumwas distinguished from A tubaeforme andwas thought to be host-specific for dogs [31 32]

To date feline hookworms (including A tubaeforme Abraziliense A ceylanicum A caninumand U stenocephala)had been reported nineteen times worldwide (Table 2) Obvi-ouslyA tubaeformewas themost common specieswhichwasreported in Australia (6 times) [6 7 11 19 21 22] USA (2times) [29 30] once in South America (Brazil) [23] Central

BioMed Research International 5

America (Costa Rica) [24] Europe (Italy Spain) [25 28] andMiddle East (Qatar) as well [27] while this parasite had notbeen reported fromcats inChina (EastAsia) [15 16]Malaysia(Southeast Asia) [26] andThailand [14]

In China A caninum was reported twice in Sichuanprovince southwest China with prevalence of 25 and 51respectively [15 16] while in our survey overall higher preva-lence (951) of A caninum infections in cats was detectedin Guangzhou (southern China) Our results suggested thatthe predominant species of hookworms in cats was Acaninum in China while A tubaeforme was considered tobe the predominant species in Australia [11] which stronglysupports our suggestion that the prevalent species is relatedto its geographical distribution as well as cats could be themain host for A caninum in China

The first cat-derived A caninum complete ITS sequences(GenBank KC755026 KC755028 and KC755029) are pre-sented in our study The length of obtained sequences(738 bp) was identical to the dog-derived A caninum ITSsequence as well as the sequence similarity range was 986ndash100 In addition A caninum from both cats and dogs wasconnected in a single cluster in the constructed phylogenetictree This finding indicated that there could be a cross-infection of A caninum between dogs and cats in the studiedareas

In conclusion the results of this study demonstratedthat The PCR-RFLP technique described in this study was arapid and straightforward method for the identification anddiscrimination of A caninum Moreover the ITS sequencescould be used to identify this hookworm species from differ-ent local hosts Current information regarding the prevalenceof A caninum showed possible cross-infections betweendifferent hosts Therefore it is imperative to have currentinformation regarding the prevalence of this hookwormand the associated risk factors of this infection This willallow a more effective implementation of strategic controlprogrammes for hookworm infections

Acknowledgments

This work was funded by the National Natural ScienceFoundation of China (Grant nos 30972179 and 31272551)The authors would like to thank Xu-hui Lin and the humaneshelterrsquo personnel for helping them in collecting all of thesamples

References

[1] L M C e Silva R R C Miranda H A Santos and E ML Rabelo ldquoDifferential diagnosis of dog hookworms based onPCR-RFLP from the ITS region of their rDNArdquo VeterinaryParasitology vol 140 no 3-4 pp 373ndash377 2006

[2] S Abubucker J Martin Y Yin et al ldquoThe canine hookwormgenome analysis and classification of Ancylostoma caninumsurvey sequencesrdquoMolecular and Biochemical Parasitology vol157 no 2 pp 187ndash192 2008

[3] M V Periago and J M Bethony ldquoHookworm virulence factorsmaking themost of the hostrdquoMicrobes and Infection vol 14 pp1451ndash1464 2012

[4] Y Yang J Zheng and J Chen ldquoCloning sequencing andphylogenetic analysis of the small GTPase gene cdc-42 fromAncylostoma caninumrdquo Experimental Parasitology vol 132 pp550ndash555 2012

[5] P Setasuban and A H Waddell ldquoHookworms in cats and dogsin Queenslandrdquo Australian Veterinary Journal vol 49 no 2 p110 1973

[6] J D Kelly and B K Y Ng ldquoHelminth parasites of dogs and catsI prevalence in urban environments in Australiardquo AustralianVeterinary Practitioner vol 5 pp 133ndash141 1975

[7] S L Wilson-Hanson and CW Prescott ldquoA survey for parasitesin catsrdquoAustralian Veterinary Journal vol 59 no 6 p 194 1982

[8] I Beveridge ldquoAustralian hookworms (Ancylostomatoidea) Areview of the species present their distributions and biogeo-graphical originsrdquo Parassitologia vol 44 no 1-2 pp 83ndash882002

[9] R B Gasser L E Stewart and R Speare ldquoGenetic markersin ribosomal DNA for hookworm identificationrdquo Acta Tropicavol 62 no 1 pp 15ndash21 1996

[10] P Prociv and J Croese ldquoHuman enteric infection with Ancy-lostoma caninum Hookworms reappraised in the light of aldquonewrdquo zoonosisrdquo Acta Tropica vol 62 no 1 pp 23ndash44 1996

[11] C S Palmer R J Traub I D Robertson et al ldquoThe veterinaryand public health significance of hookworm in dogs andcats in Australia and the status of A ceylanicumrdquo VeterinaryParasitology vol 145 no 3-4 pp 304ndash313 2007

[12] X Z Li R F Yan andXG Li ldquoInvestigation on gastrointestinalparasites of dogs inNanjingrdquoAnimal Husbandry andVeterinaryMedicine vol 43 pp 72ndash73 2011 (Chinese)

[13] J G Sun ldquoInvestigation on parasites of domestic dogs and otherlivestock in Cangxi County Sichuan Provincerdquo Chinese Journalof Zoonoses vol 3 article 53 1987 (Chinese)

[14] P Setasuban S Vajrasthira and C Muennoo ldquoPrevalenceand zoonotic potential of Ancylostoma ceylanicum in cats inThailandrdquo Southeast Asian Journal of Tropical Medicine andPublic Health vol 7 no 4 pp 534ndash539 1976

[15] M F Yang ldquoInvestigation of felid parasite in Sichuanrdquo SichuanJournal of Agricultural Science vol 2 pp 45ndash48 1987 (Chinese)

[16] Y Feng Q He and J P Li ldquoInvestigation of canine and felidparasite in TongjiangCountyrdquoModernAgricultural Sciences andTechnology vol 11 pp 354ndash355 2011 (Chinese)

[17] J DThompson T J Gibson F Plewniak F Jeanmougin andDG Higgins ldquoTheClustal X windows interface flexible strategiesformultiple sequence alignment aided by quality analysis toolsrdquoNucleic Acids Research vol 24 pp 4876ndash4882 1997

[18] G G Gregory and B L Munday ldquoInternal parasites of feralcats from the TasmanianMidlands and King IslandrdquoAustralianVeterinary Journal vol 52 no 7 pp 317ndash320 1976

[19] C W Prescott Parasitic Diseases of the Cat in Australia TheUniversity of Sydney Sydney Australia 1984

[20] E Moore and M G OrsquoCallaghan ldquoHelminths of dogs andcats determined by faecal examinations in Adelaide SouthAustraliardquo Australian Veterinary Journal vol 62 no 6 pp 198ndash199 1985

[21] B P Meloni R C AThompson R M Hopkins J A Reynold-son and M Gracey ldquoThe prevalence of Giardia and otherintestinal parasites in children dogs and cats from Aboriginalcommunities in the Kimberleyrdquo Medical Journal of Australiavol 158 no 3 pp 157ndash159 1993

[22] MGOrsquoCallaghan and I Beveridge ldquoGastro-intestinal parasitesof feral cats in the Northern Territoryrdquo Transactions of the RoyalSociety of South Australia vol 120 no 3-4 pp 175ndash176 1996

6 BioMed Research International

[23] N Labarthe M L Serrao A M R Ferreira N K O Almeidaand J Guerrero ldquoA survey of gastrointestinal helminths in catsof the metropolitan region of Rio de Janeiro Brazilrdquo VeterinaryParasitology vol 123 no 1-2 pp 133ndash139 2004

[24] AV Scorza CDuncan LMiles andMR Lappin ldquoPrevalenceof selected zoonotic and vector-borne agents in dogs and catsin Costa RicardquoVeterinary Parasitology vol 183 no 1-2 pp 178ndash183 2011

[25] F Riggio R Mannella G Ariti and S Perrucci ldquoIntestinaland lung parasites in owned dogs and cats from central ItalyrdquoVeterinary Parasitology vol 193 pp 78ndash84 2013

[26] R Ngui Y A L Lim R Traub R Mahmud and M SMistam ldquoEpidemiological and genetic data supporting thetransmission of Ancylostoma ceylanicum among human anddomestic animalsrdquo PLoS Neglected Tropical Diseases vol 6 no2 Article ID e1522 2012

[27] M A Abu-Madi J M Behnke K S Prabhaker R Al-Ibrahimand J W Lewis ldquoIntestinal helminths of feral cat populationsfrom urban and suburban districts of Qatarrdquo Veterinary Para-sitology vol 168 no 3-4 pp 284ndash292 2010

[28] C Calvete J Lucientes J A Castillo et al ldquoGastrointestinalhelminth parasites in stray cats from the mid-Ebro ValleySpainrdquo Veterinary Parasitology vol 75 no 2-3 pp 235ndash2401998

[29] T C Anderson G W Foster and D J Forrester ldquoHookwormsof feral cats in Floridardquo Veterinary Parasitology vol 115 no 1pp 19ndash24 2003

[30] M C Gates and T J Nolan ldquoEndoparasite prevalence andrecurrence across different age groups of dogs and catsrdquoVeterinary Parasitology vol 166 no 1-2 pp 153ndash158 2009

[31] E Biocca ldquoRediscrizione di Ancylostoma tubaeforme (Zedler1800) parassita del gatto considerate erroneamente sinonimodi Ancylostoma caninum (Ercolani 1859) parassita del camerdquoRivista di Parassitologia vol 15 pp 267ndash278 1954

[32] R B Burrows ldquoComparative morphology of Ancylostomatubaeforme (Zeder 1800) and Ancylostoma caninum (Ercolani1859)rdquoThe Journal of Parasitology vol 48 pp 715ndash718 1962

[33] J A Scott ldquoAn experimental study of the development of Ancy-lostoma caninum in normal and abnormal hostsrdquo AmericanJournal of Epidemiology vol 8 no 2 pp 158ndash204 1928

[34] J A Scott ldquoExperimental demonstration of a strain of the doghookworm Ancylostoma caninum especially adapted to thecatrdquo Journal for Parasitology vol 3 pp 209ndash215 1929

[35] O R Mccoy ldquoImmunity reactions of the dog against hook-worm (Ancylostoma caninum) under conditions of repeatedinfectionrdquo American Journal of Epidemiology vol 14 no 2 pp268ndash303 1931

[36] A O Foster and W W Cort ldquoThe effect of a deficient dieton the susceptibility of dogs and cats to non-specific strains ofhookwormsrdquo American Journal of Epidemiology vol 16 no 2pp 582ndash601 1932

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2 BioMed Research International

Table 1 Predictive restriction patterns by endonucleases EcoR II BsuR I and Taq I at ITS1 and 58S locus

Species PCR amplicon (bp) Cleavage site Predicted fragment size (bp)EcoR II BsuR I Taq I

A caninum 404 minus minus 3+ 60307 (12U 25U)A tubaeforme 405 minus minus 2+ 60333 (12U)A ceylanicum 404 + minus 76328A braziliense 408 2+ minus 76122210U stenocephala 406 minus + 87319UMeans that the fragment is too small to visible

heavy monsoon rains It covers an area of approximately8000 km2 divided into 10 geographical districts with anestimated population of approximately 12 million This citycontains a large number of sheltered cats and owns twolocal humane shelters for stray cats in Conghua and Baiyundistricts

22 Fecal Sample Collection and Processing Cat fecal samples(119899 = 102) were collected from Conghua (119899 = 72) and Baiyun(119899 = 30) humane shelters during March and July 2012 Asingle sample was collected in clean container from eachcat directly transported to laboratory preserved in 25potassium dichromate and stored at 4∘C To detect thepresence of hookworm eggs direct microscopic examinationwas done by saturated sodium chloride and glucose flotationPositive fecal samples were further characterized by molecu-lar procedures

23 Genomic DNA Extraction DNAs were extracted directlyfrom fecal samples using a commercial DNA extraction kit(QIAamp DNA Stool Mini Kit QIAgen Hilden Germany)according to the manufacturerrsquos protocols However allsamples were pretreated with 5 cycles of heating at 100∘Cfor 5 minutes followed by immediate freezing at minus80∘Cfor 5 minutes A negative control (water) was used in eachextraction group Extracted DNAs were then stored at minus20∘C

24 Primers and Restriction Enzyme One pair of primersAF (51015840-CTTTGTCGGGAAGGTTGG-31015840) and AR (51015840-TTC-ACCACTCTAAGCGTCT-31015840) was designed from conservedregion of ITS sequences of five species of hookwormsincluding A caninum (AM85010 AM850105) A tubae-forme (JQ812691) A ceylanicum (DQ381541 DQ780009)A braziliense (DQ359149 DQ438056) and U stenocephala(HQ262053 AF194145) by Primer Premier 50 to amplify404 bp region ofA caninum 405 bp ofA tubaeforme 408 bpof A braziliense 404 bp of A ceylanicum and 406 bp of Ustenocephalawhich contain ITS1 and 58S rRNA regionsThefive species of hookworms could be distinguished by restric-tion endonucleases EcoRII BsuRI and Taq I for differentcutting sites on the sequence according to the analysis byPrimer Premier 50 The theoretical cutting patterns of thefive different hookworm DNA fragments treated by threerestriction enzymes are shown in Table 1 EcoR II couldidentify A ceylanicum and A braziliense while BsuRI canonly identify U stenocephala and Taq I was distinguishedbetween A caninum and A tubaeforme

Another pair of primers CAF (51015840-GACTGCGGACTG-CTGTAT-31015840) and CAR (51015840-AAGTTCAGCGGGTAGTCA-31015840) was designed by Primer Premier 50 based on ITSsequences (JQ812694 AJ920347 and AM039739) of A can-inum to amplify the complete ITS sequence of cat-derived Acaninum

25 PCR-RFLP Both PCRs were performed in 25 120583L volumecontaining 2 120583L of the DNA sample 02120583L ofTaq polymerase(TaKaRa Dalian China) 25 120583L of 10timesTaq buffer (TaKaRa)2 120583L of diethylnitrophenyl thiophosphate (dNTP TaKaRa)mixture 05 120583L of each primer (AFAR or CAFCAR50mM) and 173 120583L of distilled water PCR cycling param-eters were as follows 1 cycle at 96∘C for 5 minutes then 35cycles of 96∘C for 30 seconds at 60∘C for 30 seconds and at72∘C for 90 seconds followed by 1 cycle at 72∘C for 7minutes

RFLP analysis was performed by digesting 7 120583L of PCRproduct with 2U of each restriction endonuclease (TaKaRa)in a final volume of 20120583L for 3 hours at 37∘C PCR productsand restriction fragments were analyzed after electrophoresisin 2 and 3 agarose gels with 02 120583gmL of ethidium bro-mide staining and were visualized on a UV transilluminator

26 Sequence Confirmation and Phylogenetic Analysis Posi-tive amplicons were purified and sequenced using ABI 3730automated DNA sequencer (BigDye Terminator Chemistry)Obtained sequences were aligned with 15 ITS referencesequences using Clustal X programs [17] Phylogenetic treeswere constructed usingMEGA version 51 (MEGA51Molec-ular Evolutionary Genetics Analysis software Arizona StateUniversity Tempe Arizona USA) Bootstrap analyses wereconducted using 1000 replicates to assess the reliability ofinferred tree topologies Neighbor-joining algorithms wereconducted using the Kimura 2 parameter distance analysisObtained nucleotide sequences have been deposited in theGenBank database under accession numbers KC755015 andKC755025

3 Results

Of the 102 collected fecal samples 85 samples (833) weremicroscopically positive while 97 samples (951) were PCRpositive for hookwormThe prevalence of A caninum in catsfrom suburban area (861 Conghua) was higher than thatfrom urban area (767 Baiyun)

The results showed that U stenocephala was absent in the97 examined PCR samples where there was no enzymaticdigestion by restriction endonucleases BsuRI (not shown)

BioMed Research International 3

Figure 1 PCR amplicons digested by restriction endonucleases EcoR II Lanes 1ndash62 samples from Conghua district lanes 62ndash85 samplesfrom Baiyun district lanes 86ndash97 microscopically negative samples M DL-2000 DNA marker

Figure 2 PCR amplicons digested by restriction endonucleases Taq I Lanes 1ndash53 random samples from 56 samples P PCR amplicon MDL-2000 DNA marker

Cat-China-KC755025Cat-China-KC755022Dog-China-A caninum-AM850106Dog-China-A caninum-EU159416Cat-China-KC755018

50 Dog-China-A caninum-EU159415Dog-China-A caninum-AM850105

Cat-China-KC755023Dog-America-A caninum-JQ812694

60 Cat-China-KC755019Cat-China-KC755024

42 Cat-China-KC75501751 35 Cat-China-KC755016

Human-China-A duodenale-EU344797Cat-America-A tubaeforme-JQ812691

Dog-America-A braziliense-JQ81269259 Cat-America-A braziliense-JQ812693

Cat-China-KC755020Dog-India-A ceylanicum-DQ780009

77 Dog-America-A ceylanicum-DQ381541Cat-China-KC755021Cat-China-KC755015

Urocyon-USA-U stenocephala-HQ262053Urocyon-USA-U stenocephala-HQ262054

92

64

9694

35

87

100

663370

100

0005

A braziliense

U stenocephala

A caninum

A ceylanicum

Figure 3 Phylogenetic tree of hookworm isolates based on the ITS1 and 58S rRNA sequences with neighbor-joining algorithm using Kimuratwo-parameter The reference sequences are available in the GenBank by their accession numbers The isolates of this study are shown in redcolor

Figure 1 shows the digestion results of the 97 PCR ampliconsby restriction endonucleases EcoR II in which 41 samplesshowed three bands revealing that these samples weremixed infections with A ceylanicum and one undeterminedhookworm (A caninum or other) while 56 samples showedone band revealing that these samples were single infectionwith one undetermined hookworm (A caninum or other)Fifty three samples from the 56 single infection samples wererandomly chosen for the Taq I digestion the results showthat all these samples were infected with only one species(Figure 2)Thereafter 11 positive samples (656 and 541)weresuccessfully sequenced the phylogenetic tree based on those

sequences with 15 reference sequences (Figure 3) revealedthat the 56 positive samples were infected with A caninumwhile the 41 samples were infected with both A ceylanicumand A caninum without any A tubaeforme infection in ourstudy Thus the overall prevalence of A caninum infectionwas 951 with a prevalence of 549 single infection and402 mixed infections with A ceylanicum

Three complete ITS sequences from cat-derived A can-inum isolates (M45 M63 and M84) were obtained byPrimer CAFCAR and submitted in the GenBank underaccession numbers (KC755026 KC755028 and KC755029)All ITS sequences were 738 bp in length which was the same

4 BioMed Research International

Table 2 Previous investigations of hookworm species in cats worldwideCountry (area) Detection method Species Prevalence Source of animal Reference

Australia (Sydney) Necropsy A tubaeforme 35 Pound [6]U stenocephala 05 (119899 = 404)

Australia (Tasmania) Necropsy U stenocephala 2 (119899 = 86) Feral cats [18]Australia (Brsibane) Necropsy A tubaeforme 19 (119899 = 404) Pound [7]Australia (Brisbane) Necropsy A tubaeforme 81 Pound [19]Australia (Adelaide) Fecal exam U stenocephala 03 (119899 = 376) Vet practice [20]Australia (Kimberley) Necropsy A tubaeforme 20 (119899 = 34) Aboriginal community [21]Australia (Northern territory) Necropsy A tubaeforme 13 Feral cats [22]

Australia (countrywide) PCR-RFLP A tubaeforme 70 (119899 = 10) Refuge and pet cats [11]A caninum 30 (119899 = 10)

Brazil (Rio de Janeiro) Necropsy A braziliense 659 (119899 = 135) Cat (shelters and ZoonosesControl Center)

[23]A tubaeforme 89 (119899 = 135)

China (Sichuan) Necropsy

A caninum 25 (119899 = 20)

Domestic cat [15]A braziliense 10 (119899 = 20)A ceylanicum 5 (119899 = 20)U stenocephala 10 (119899 = 20)

China (Sichuan) NecropsyA caninum 51 (119899 = 149)

Domestic cat [16]A braziliense 174 (119899 = 149)U stenocephala 14 (119899 = 149)

Costa Rica (San Isidro ELGeneral) Fecal exam A tubaeforme 11 (119899 = 9) Refuge cat [24]

Thailand (Prachin Buri) A ceylanicumA caninum

9223 Cat [14]

Italy (central) Larvae (L3) exam A tubaeforme 12 (119899 = 81) Cat (pet) [25]U stenocephala 37 (119899 = 81)

Malaysia (West Malaysia) PCR-sequencing A ceylanicumA braziliense

261 (119899 = 23)43 (119899 = 23) Domestic cat [26]

Qatar (Doha) Necropsy A tubaeforme 147 (119899 = 658) Cat (feral) [27]Spain (mid-Ebro Valley) Necropsy A tubaeforme 293 (119899 = 58) Cat (stray) [28]

USA (Florida) Necropsy A tubaeforme 75 (119899 = 60) Cat (feral) [29]A braziliense 33 (119899 = 60)

USA (Pennsylvania) Fecal exam A tubaeforme 05 (119899 = 1566) Cat (pet) [30]

length presented by the A caninum ITS region (AM850105AM850106 EU159415 and EU159416) isolated from dogs inthis area DNA sequences were assembled using DNAStar(version 7 Madison WI USA) and multiple-sequence align-ment was performed with MegAlign program Comparativeanalysis of the three complete ITS sequences obtained fromcat-derived A caninum showed the same length (738 bp) ofdog-derived A caninum with a similarity of 992ndash997However compared to the ITS sequences of dog-derivedA caninum isolates (AM850105 AM850106 EU159415 andEU159416) the sequence variation range was 986ndash100where only one cat isolate (M63) showed 100 sequencesimilarity compared with two dog-derived A caninum iso-lates (AM850106 and EU159416) in the same studied areaThe sequence similarities of the cat-derived A caninum withA ceylanicum (KC755027) A tubaeforme (JQ812691) and Abraziliense (JQ812692) were 970ndash973 973ndash976 and916ndash921 respectively

4 Discussion

As early as the 19th century hookworms in cats and dogshad been described by Zedler (1800) and Ercolani (1859)[31] For nearly 100 years the common hookworm of bothdogs and cats was referred as Ancylostoma caninum [32]Later then some authors stated that it was difficult to infectdogs with larvae from cats and vice versa [32ndash36] Studiesin Europe Africa and Australia described themorphologicaldifferences of hookworms obtained from dogs and catswhere A caninumwas distinguished from A tubaeforme andwas thought to be host-specific for dogs [31 32]

To date feline hookworms (including A tubaeforme Abraziliense A ceylanicum A caninumand U stenocephala)had been reported nineteen times worldwide (Table 2) Obvi-ouslyA tubaeformewas themost common specieswhichwasreported in Australia (6 times) [6 7 11 19 21 22] USA (2times) [29 30] once in South America (Brazil) [23] Central

BioMed Research International 5

America (Costa Rica) [24] Europe (Italy Spain) [25 28] andMiddle East (Qatar) as well [27] while this parasite had notbeen reported fromcats inChina (EastAsia) [15 16]Malaysia(Southeast Asia) [26] andThailand [14]

In China A caninum was reported twice in Sichuanprovince southwest China with prevalence of 25 and 51respectively [15 16] while in our survey overall higher preva-lence (951) of A caninum infections in cats was detectedin Guangzhou (southern China) Our results suggested thatthe predominant species of hookworms in cats was Acaninum in China while A tubaeforme was considered tobe the predominant species in Australia [11] which stronglysupports our suggestion that the prevalent species is relatedto its geographical distribution as well as cats could be themain host for A caninum in China

The first cat-derived A caninum complete ITS sequences(GenBank KC755026 KC755028 and KC755029) are pre-sented in our study The length of obtained sequences(738 bp) was identical to the dog-derived A caninum ITSsequence as well as the sequence similarity range was 986ndash100 In addition A caninum from both cats and dogs wasconnected in a single cluster in the constructed phylogenetictree This finding indicated that there could be a cross-infection of A caninum between dogs and cats in the studiedareas

In conclusion the results of this study demonstratedthat The PCR-RFLP technique described in this study was arapid and straightforward method for the identification anddiscrimination of A caninum Moreover the ITS sequencescould be used to identify this hookworm species from differ-ent local hosts Current information regarding the prevalenceof A caninum showed possible cross-infections betweendifferent hosts Therefore it is imperative to have currentinformation regarding the prevalence of this hookwormand the associated risk factors of this infection This willallow a more effective implementation of strategic controlprogrammes for hookworm infections

Acknowledgments

This work was funded by the National Natural ScienceFoundation of China (Grant nos 30972179 and 31272551)The authors would like to thank Xu-hui Lin and the humaneshelterrsquo personnel for helping them in collecting all of thesamples

References

[1] L M C e Silva R R C Miranda H A Santos and E ML Rabelo ldquoDifferential diagnosis of dog hookworms based onPCR-RFLP from the ITS region of their rDNArdquo VeterinaryParasitology vol 140 no 3-4 pp 373ndash377 2006

[2] S Abubucker J Martin Y Yin et al ldquoThe canine hookwormgenome analysis and classification of Ancylostoma caninumsurvey sequencesrdquoMolecular and Biochemical Parasitology vol157 no 2 pp 187ndash192 2008

[3] M V Periago and J M Bethony ldquoHookworm virulence factorsmaking themost of the hostrdquoMicrobes and Infection vol 14 pp1451ndash1464 2012

[4] Y Yang J Zheng and J Chen ldquoCloning sequencing andphylogenetic analysis of the small GTPase gene cdc-42 fromAncylostoma caninumrdquo Experimental Parasitology vol 132 pp550ndash555 2012

[5] P Setasuban and A H Waddell ldquoHookworms in cats and dogsin Queenslandrdquo Australian Veterinary Journal vol 49 no 2 p110 1973

[6] J D Kelly and B K Y Ng ldquoHelminth parasites of dogs and catsI prevalence in urban environments in Australiardquo AustralianVeterinary Practitioner vol 5 pp 133ndash141 1975

[7] S L Wilson-Hanson and CW Prescott ldquoA survey for parasitesin catsrdquoAustralian Veterinary Journal vol 59 no 6 p 194 1982

[8] I Beveridge ldquoAustralian hookworms (Ancylostomatoidea) Areview of the species present their distributions and biogeo-graphical originsrdquo Parassitologia vol 44 no 1-2 pp 83ndash882002

[9] R B Gasser L E Stewart and R Speare ldquoGenetic markersin ribosomal DNA for hookworm identificationrdquo Acta Tropicavol 62 no 1 pp 15ndash21 1996

[10] P Prociv and J Croese ldquoHuman enteric infection with Ancy-lostoma caninum Hookworms reappraised in the light of aldquonewrdquo zoonosisrdquo Acta Tropica vol 62 no 1 pp 23ndash44 1996

[11] C S Palmer R J Traub I D Robertson et al ldquoThe veterinaryand public health significance of hookworm in dogs andcats in Australia and the status of A ceylanicumrdquo VeterinaryParasitology vol 145 no 3-4 pp 304ndash313 2007

[12] X Z Li R F Yan andXG Li ldquoInvestigation on gastrointestinalparasites of dogs inNanjingrdquoAnimal Husbandry andVeterinaryMedicine vol 43 pp 72ndash73 2011 (Chinese)

[13] J G Sun ldquoInvestigation on parasites of domestic dogs and otherlivestock in Cangxi County Sichuan Provincerdquo Chinese Journalof Zoonoses vol 3 article 53 1987 (Chinese)

[14] P Setasuban S Vajrasthira and C Muennoo ldquoPrevalenceand zoonotic potential of Ancylostoma ceylanicum in cats inThailandrdquo Southeast Asian Journal of Tropical Medicine andPublic Health vol 7 no 4 pp 534ndash539 1976

[15] M F Yang ldquoInvestigation of felid parasite in Sichuanrdquo SichuanJournal of Agricultural Science vol 2 pp 45ndash48 1987 (Chinese)

[16] Y Feng Q He and J P Li ldquoInvestigation of canine and felidparasite in TongjiangCountyrdquoModernAgricultural Sciences andTechnology vol 11 pp 354ndash355 2011 (Chinese)

[17] J DThompson T J Gibson F Plewniak F Jeanmougin andDG Higgins ldquoTheClustal X windows interface flexible strategiesformultiple sequence alignment aided by quality analysis toolsrdquoNucleic Acids Research vol 24 pp 4876ndash4882 1997

[18] G G Gregory and B L Munday ldquoInternal parasites of feralcats from the TasmanianMidlands and King IslandrdquoAustralianVeterinary Journal vol 52 no 7 pp 317ndash320 1976

[19] C W Prescott Parasitic Diseases of the Cat in Australia TheUniversity of Sydney Sydney Australia 1984

[20] E Moore and M G OrsquoCallaghan ldquoHelminths of dogs andcats determined by faecal examinations in Adelaide SouthAustraliardquo Australian Veterinary Journal vol 62 no 6 pp 198ndash199 1985

[21] B P Meloni R C AThompson R M Hopkins J A Reynold-son and M Gracey ldquoThe prevalence of Giardia and otherintestinal parasites in children dogs and cats from Aboriginalcommunities in the Kimberleyrdquo Medical Journal of Australiavol 158 no 3 pp 157ndash159 1993

[22] MGOrsquoCallaghan and I Beveridge ldquoGastro-intestinal parasitesof feral cats in the Northern Territoryrdquo Transactions of the RoyalSociety of South Australia vol 120 no 3-4 pp 175ndash176 1996

6 BioMed Research International

[23] N Labarthe M L Serrao A M R Ferreira N K O Almeidaand J Guerrero ldquoA survey of gastrointestinal helminths in catsof the metropolitan region of Rio de Janeiro Brazilrdquo VeterinaryParasitology vol 123 no 1-2 pp 133ndash139 2004

[24] AV Scorza CDuncan LMiles andMR Lappin ldquoPrevalenceof selected zoonotic and vector-borne agents in dogs and catsin Costa RicardquoVeterinary Parasitology vol 183 no 1-2 pp 178ndash183 2011

[25] F Riggio R Mannella G Ariti and S Perrucci ldquoIntestinaland lung parasites in owned dogs and cats from central ItalyrdquoVeterinary Parasitology vol 193 pp 78ndash84 2013

[26] R Ngui Y A L Lim R Traub R Mahmud and M SMistam ldquoEpidemiological and genetic data supporting thetransmission of Ancylostoma ceylanicum among human anddomestic animalsrdquo PLoS Neglected Tropical Diseases vol 6 no2 Article ID e1522 2012

[27] M A Abu-Madi J M Behnke K S Prabhaker R Al-Ibrahimand J W Lewis ldquoIntestinal helminths of feral cat populationsfrom urban and suburban districts of Qatarrdquo Veterinary Para-sitology vol 168 no 3-4 pp 284ndash292 2010

[28] C Calvete J Lucientes J A Castillo et al ldquoGastrointestinalhelminth parasites in stray cats from the mid-Ebro ValleySpainrdquo Veterinary Parasitology vol 75 no 2-3 pp 235ndash2401998

[29] T C Anderson G W Foster and D J Forrester ldquoHookwormsof feral cats in Floridardquo Veterinary Parasitology vol 115 no 1pp 19ndash24 2003

[30] M C Gates and T J Nolan ldquoEndoparasite prevalence andrecurrence across different age groups of dogs and catsrdquoVeterinary Parasitology vol 166 no 1-2 pp 153ndash158 2009

[31] E Biocca ldquoRediscrizione di Ancylostoma tubaeforme (Zedler1800) parassita del gatto considerate erroneamente sinonimodi Ancylostoma caninum (Ercolani 1859) parassita del camerdquoRivista di Parassitologia vol 15 pp 267ndash278 1954

[32] R B Burrows ldquoComparative morphology of Ancylostomatubaeforme (Zeder 1800) and Ancylostoma caninum (Ercolani1859)rdquoThe Journal of Parasitology vol 48 pp 715ndash718 1962

[33] J A Scott ldquoAn experimental study of the development of Ancy-lostoma caninum in normal and abnormal hostsrdquo AmericanJournal of Epidemiology vol 8 no 2 pp 158ndash204 1928

[34] J A Scott ldquoExperimental demonstration of a strain of the doghookworm Ancylostoma caninum especially adapted to thecatrdquo Journal for Parasitology vol 3 pp 209ndash215 1929

[35] O R Mccoy ldquoImmunity reactions of the dog against hook-worm (Ancylostoma caninum) under conditions of repeatedinfectionrdquo American Journal of Epidemiology vol 14 no 2 pp268ndash303 1931

[36] A O Foster and W W Cort ldquoThe effect of a deficient dieton the susceptibility of dogs and cats to non-specific strains ofhookwormsrdquo American Journal of Epidemiology vol 16 no 2pp 582ndash601 1932

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

Figure 1 PCR amplicons digested by restriction endonucleases EcoR II Lanes 1ndash62 samples from Conghua district lanes 62ndash85 samplesfrom Baiyun district lanes 86ndash97 microscopically negative samples M DL-2000 DNA marker

Figure 2 PCR amplicons digested by restriction endonucleases Taq I Lanes 1ndash53 random samples from 56 samples P PCR amplicon MDL-2000 DNA marker

Cat-China-KC755025Cat-China-KC755022Dog-China-A caninum-AM850106Dog-China-A caninum-EU159416Cat-China-KC755018

50 Dog-China-A caninum-EU159415Dog-China-A caninum-AM850105

Cat-China-KC755023Dog-America-A caninum-JQ812694

60 Cat-China-KC755019Cat-China-KC755024

42 Cat-China-KC75501751 35 Cat-China-KC755016

Human-China-A duodenale-EU344797Cat-America-A tubaeforme-JQ812691

Dog-America-A braziliense-JQ81269259 Cat-America-A braziliense-JQ812693

Cat-China-KC755020Dog-India-A ceylanicum-DQ780009

77 Dog-America-A ceylanicum-DQ381541Cat-China-KC755021Cat-China-KC755015

Urocyon-USA-U stenocephala-HQ262053Urocyon-USA-U stenocephala-HQ262054

92

64

9694

35

87

100

663370

100

0005

A braziliense

U stenocephala

A caninum

A ceylanicum

Figure 3 Phylogenetic tree of hookworm isolates based on the ITS1 and 58S rRNA sequences with neighbor-joining algorithm using Kimuratwo-parameter The reference sequences are available in the GenBank by their accession numbers The isolates of this study are shown in redcolor

Figure 1 shows the digestion results of the 97 PCR ampliconsby restriction endonucleases EcoR II in which 41 samplesshowed three bands revealing that these samples weremixed infections with A ceylanicum and one undeterminedhookworm (A caninum or other) while 56 samples showedone band revealing that these samples were single infectionwith one undetermined hookworm (A caninum or other)Fifty three samples from the 56 single infection samples wererandomly chosen for the Taq I digestion the results showthat all these samples were infected with only one species(Figure 2)Thereafter 11 positive samples (656 and 541)weresuccessfully sequenced the phylogenetic tree based on those

sequences with 15 reference sequences (Figure 3) revealedthat the 56 positive samples were infected with A caninumwhile the 41 samples were infected with both A ceylanicumand A caninum without any A tubaeforme infection in ourstudy Thus the overall prevalence of A caninum infectionwas 951 with a prevalence of 549 single infection and402 mixed infections with A ceylanicum

Three complete ITS sequences from cat-derived A can-inum isolates (M45 M63 and M84) were obtained byPrimer CAFCAR and submitted in the GenBank underaccession numbers (KC755026 KC755028 and KC755029)All ITS sequences were 738 bp in length which was the same

4 BioMed Research International

Table 2 Previous investigations of hookworm species in cats worldwideCountry (area) Detection method Species Prevalence Source of animal Reference

Australia (Sydney) Necropsy A tubaeforme 35 Pound [6]U stenocephala 05 (119899 = 404)

Australia (Tasmania) Necropsy U stenocephala 2 (119899 = 86) Feral cats [18]Australia (Brsibane) Necropsy A tubaeforme 19 (119899 = 404) Pound [7]Australia (Brisbane) Necropsy A tubaeforme 81 Pound [19]Australia (Adelaide) Fecal exam U stenocephala 03 (119899 = 376) Vet practice [20]Australia (Kimberley) Necropsy A tubaeforme 20 (119899 = 34) Aboriginal community [21]Australia (Northern territory) Necropsy A tubaeforme 13 Feral cats [22]

Australia (countrywide) PCR-RFLP A tubaeforme 70 (119899 = 10) Refuge and pet cats [11]A caninum 30 (119899 = 10)

Brazil (Rio de Janeiro) Necropsy A braziliense 659 (119899 = 135) Cat (shelters and ZoonosesControl Center)

[23]A tubaeforme 89 (119899 = 135)

China (Sichuan) Necropsy

A caninum 25 (119899 = 20)

Domestic cat [15]A braziliense 10 (119899 = 20)A ceylanicum 5 (119899 = 20)U stenocephala 10 (119899 = 20)

China (Sichuan) NecropsyA caninum 51 (119899 = 149)

Domestic cat [16]A braziliense 174 (119899 = 149)U stenocephala 14 (119899 = 149)

Costa Rica (San Isidro ELGeneral) Fecal exam A tubaeforme 11 (119899 = 9) Refuge cat [24]

Thailand (Prachin Buri) A ceylanicumA caninum

9223 Cat [14]

Italy (central) Larvae (L3) exam A tubaeforme 12 (119899 = 81) Cat (pet) [25]U stenocephala 37 (119899 = 81)

Malaysia (West Malaysia) PCR-sequencing A ceylanicumA braziliense

261 (119899 = 23)43 (119899 = 23) Domestic cat [26]

Qatar (Doha) Necropsy A tubaeforme 147 (119899 = 658) Cat (feral) [27]Spain (mid-Ebro Valley) Necropsy A tubaeforme 293 (119899 = 58) Cat (stray) [28]

USA (Florida) Necropsy A tubaeforme 75 (119899 = 60) Cat (feral) [29]A braziliense 33 (119899 = 60)

USA (Pennsylvania) Fecal exam A tubaeforme 05 (119899 = 1566) Cat (pet) [30]

length presented by the A caninum ITS region (AM850105AM850106 EU159415 and EU159416) isolated from dogs inthis area DNA sequences were assembled using DNAStar(version 7 Madison WI USA) and multiple-sequence align-ment was performed with MegAlign program Comparativeanalysis of the three complete ITS sequences obtained fromcat-derived A caninum showed the same length (738 bp) ofdog-derived A caninum with a similarity of 992ndash997However compared to the ITS sequences of dog-derivedA caninum isolates (AM850105 AM850106 EU159415 andEU159416) the sequence variation range was 986ndash100where only one cat isolate (M63) showed 100 sequencesimilarity compared with two dog-derived A caninum iso-lates (AM850106 and EU159416) in the same studied areaThe sequence similarities of the cat-derived A caninum withA ceylanicum (KC755027) A tubaeforme (JQ812691) and Abraziliense (JQ812692) were 970ndash973 973ndash976 and916ndash921 respectively

4 Discussion

As early as the 19th century hookworms in cats and dogshad been described by Zedler (1800) and Ercolani (1859)[31] For nearly 100 years the common hookworm of bothdogs and cats was referred as Ancylostoma caninum [32]Later then some authors stated that it was difficult to infectdogs with larvae from cats and vice versa [32ndash36] Studiesin Europe Africa and Australia described themorphologicaldifferences of hookworms obtained from dogs and catswhere A caninumwas distinguished from A tubaeforme andwas thought to be host-specific for dogs [31 32]

To date feline hookworms (including A tubaeforme Abraziliense A ceylanicum A caninumand U stenocephala)had been reported nineteen times worldwide (Table 2) Obvi-ouslyA tubaeformewas themost common specieswhichwasreported in Australia (6 times) [6 7 11 19 21 22] USA (2times) [29 30] once in South America (Brazil) [23] Central

BioMed Research International 5

America (Costa Rica) [24] Europe (Italy Spain) [25 28] andMiddle East (Qatar) as well [27] while this parasite had notbeen reported fromcats inChina (EastAsia) [15 16]Malaysia(Southeast Asia) [26] andThailand [14]

In China A caninum was reported twice in Sichuanprovince southwest China with prevalence of 25 and 51respectively [15 16] while in our survey overall higher preva-lence (951) of A caninum infections in cats was detectedin Guangzhou (southern China) Our results suggested thatthe predominant species of hookworms in cats was Acaninum in China while A tubaeforme was considered tobe the predominant species in Australia [11] which stronglysupports our suggestion that the prevalent species is relatedto its geographical distribution as well as cats could be themain host for A caninum in China

The first cat-derived A caninum complete ITS sequences(GenBank KC755026 KC755028 and KC755029) are pre-sented in our study The length of obtained sequences(738 bp) was identical to the dog-derived A caninum ITSsequence as well as the sequence similarity range was 986ndash100 In addition A caninum from both cats and dogs wasconnected in a single cluster in the constructed phylogenetictree This finding indicated that there could be a cross-infection of A caninum between dogs and cats in the studiedareas

In conclusion the results of this study demonstratedthat The PCR-RFLP technique described in this study was arapid and straightforward method for the identification anddiscrimination of A caninum Moreover the ITS sequencescould be used to identify this hookworm species from differ-ent local hosts Current information regarding the prevalenceof A caninum showed possible cross-infections betweendifferent hosts Therefore it is imperative to have currentinformation regarding the prevalence of this hookwormand the associated risk factors of this infection This willallow a more effective implementation of strategic controlprogrammes for hookworm infections

Acknowledgments

This work was funded by the National Natural ScienceFoundation of China (Grant nos 30972179 and 31272551)The authors would like to thank Xu-hui Lin and the humaneshelterrsquo personnel for helping them in collecting all of thesamples

References

[1] L M C e Silva R R C Miranda H A Santos and E ML Rabelo ldquoDifferential diagnosis of dog hookworms based onPCR-RFLP from the ITS region of their rDNArdquo VeterinaryParasitology vol 140 no 3-4 pp 373ndash377 2006

[2] S Abubucker J Martin Y Yin et al ldquoThe canine hookwormgenome analysis and classification of Ancylostoma caninumsurvey sequencesrdquoMolecular and Biochemical Parasitology vol157 no 2 pp 187ndash192 2008

[3] M V Periago and J M Bethony ldquoHookworm virulence factorsmaking themost of the hostrdquoMicrobes and Infection vol 14 pp1451ndash1464 2012

[4] Y Yang J Zheng and J Chen ldquoCloning sequencing andphylogenetic analysis of the small GTPase gene cdc-42 fromAncylostoma caninumrdquo Experimental Parasitology vol 132 pp550ndash555 2012

[5] P Setasuban and A H Waddell ldquoHookworms in cats and dogsin Queenslandrdquo Australian Veterinary Journal vol 49 no 2 p110 1973

[6] J D Kelly and B K Y Ng ldquoHelminth parasites of dogs and catsI prevalence in urban environments in Australiardquo AustralianVeterinary Practitioner vol 5 pp 133ndash141 1975

[7] S L Wilson-Hanson and CW Prescott ldquoA survey for parasitesin catsrdquoAustralian Veterinary Journal vol 59 no 6 p 194 1982

[8] I Beveridge ldquoAustralian hookworms (Ancylostomatoidea) Areview of the species present their distributions and biogeo-graphical originsrdquo Parassitologia vol 44 no 1-2 pp 83ndash882002

[9] R B Gasser L E Stewart and R Speare ldquoGenetic markersin ribosomal DNA for hookworm identificationrdquo Acta Tropicavol 62 no 1 pp 15ndash21 1996

[10] P Prociv and J Croese ldquoHuman enteric infection with Ancy-lostoma caninum Hookworms reappraised in the light of aldquonewrdquo zoonosisrdquo Acta Tropica vol 62 no 1 pp 23ndash44 1996

[11] C S Palmer R J Traub I D Robertson et al ldquoThe veterinaryand public health significance of hookworm in dogs andcats in Australia and the status of A ceylanicumrdquo VeterinaryParasitology vol 145 no 3-4 pp 304ndash313 2007

[12] X Z Li R F Yan andXG Li ldquoInvestigation on gastrointestinalparasites of dogs inNanjingrdquoAnimal Husbandry andVeterinaryMedicine vol 43 pp 72ndash73 2011 (Chinese)

[13] J G Sun ldquoInvestigation on parasites of domestic dogs and otherlivestock in Cangxi County Sichuan Provincerdquo Chinese Journalof Zoonoses vol 3 article 53 1987 (Chinese)

[14] P Setasuban S Vajrasthira and C Muennoo ldquoPrevalenceand zoonotic potential of Ancylostoma ceylanicum in cats inThailandrdquo Southeast Asian Journal of Tropical Medicine andPublic Health vol 7 no 4 pp 534ndash539 1976

[15] M F Yang ldquoInvestigation of felid parasite in Sichuanrdquo SichuanJournal of Agricultural Science vol 2 pp 45ndash48 1987 (Chinese)

[16] Y Feng Q He and J P Li ldquoInvestigation of canine and felidparasite in TongjiangCountyrdquoModernAgricultural Sciences andTechnology vol 11 pp 354ndash355 2011 (Chinese)

[17] J DThompson T J Gibson F Plewniak F Jeanmougin andDG Higgins ldquoTheClustal X windows interface flexible strategiesformultiple sequence alignment aided by quality analysis toolsrdquoNucleic Acids Research vol 24 pp 4876ndash4882 1997

[18] G G Gregory and B L Munday ldquoInternal parasites of feralcats from the TasmanianMidlands and King IslandrdquoAustralianVeterinary Journal vol 52 no 7 pp 317ndash320 1976

[19] C W Prescott Parasitic Diseases of the Cat in Australia TheUniversity of Sydney Sydney Australia 1984

[20] E Moore and M G OrsquoCallaghan ldquoHelminths of dogs andcats determined by faecal examinations in Adelaide SouthAustraliardquo Australian Veterinary Journal vol 62 no 6 pp 198ndash199 1985

[21] B P Meloni R C AThompson R M Hopkins J A Reynold-son and M Gracey ldquoThe prevalence of Giardia and otherintestinal parasites in children dogs and cats from Aboriginalcommunities in the Kimberleyrdquo Medical Journal of Australiavol 158 no 3 pp 157ndash159 1993

[22] MGOrsquoCallaghan and I Beveridge ldquoGastro-intestinal parasitesof feral cats in the Northern Territoryrdquo Transactions of the RoyalSociety of South Australia vol 120 no 3-4 pp 175ndash176 1996

6 BioMed Research International

[23] N Labarthe M L Serrao A M R Ferreira N K O Almeidaand J Guerrero ldquoA survey of gastrointestinal helminths in catsof the metropolitan region of Rio de Janeiro Brazilrdquo VeterinaryParasitology vol 123 no 1-2 pp 133ndash139 2004

[24] AV Scorza CDuncan LMiles andMR Lappin ldquoPrevalenceof selected zoonotic and vector-borne agents in dogs and catsin Costa RicardquoVeterinary Parasitology vol 183 no 1-2 pp 178ndash183 2011

[25] F Riggio R Mannella G Ariti and S Perrucci ldquoIntestinaland lung parasites in owned dogs and cats from central ItalyrdquoVeterinary Parasitology vol 193 pp 78ndash84 2013

[26] R Ngui Y A L Lim R Traub R Mahmud and M SMistam ldquoEpidemiological and genetic data supporting thetransmission of Ancylostoma ceylanicum among human anddomestic animalsrdquo PLoS Neglected Tropical Diseases vol 6 no2 Article ID e1522 2012

[27] M A Abu-Madi J M Behnke K S Prabhaker R Al-Ibrahimand J W Lewis ldquoIntestinal helminths of feral cat populationsfrom urban and suburban districts of Qatarrdquo Veterinary Para-sitology vol 168 no 3-4 pp 284ndash292 2010

[28] C Calvete J Lucientes J A Castillo et al ldquoGastrointestinalhelminth parasites in stray cats from the mid-Ebro ValleySpainrdquo Veterinary Parasitology vol 75 no 2-3 pp 235ndash2401998

[29] T C Anderson G W Foster and D J Forrester ldquoHookwormsof feral cats in Floridardquo Veterinary Parasitology vol 115 no 1pp 19ndash24 2003

[30] M C Gates and T J Nolan ldquoEndoparasite prevalence andrecurrence across different age groups of dogs and catsrdquoVeterinary Parasitology vol 166 no 1-2 pp 153ndash158 2009

[31] E Biocca ldquoRediscrizione di Ancylostoma tubaeforme (Zedler1800) parassita del gatto considerate erroneamente sinonimodi Ancylostoma caninum (Ercolani 1859) parassita del camerdquoRivista di Parassitologia vol 15 pp 267ndash278 1954

[32] R B Burrows ldquoComparative morphology of Ancylostomatubaeforme (Zeder 1800) and Ancylostoma caninum (Ercolani1859)rdquoThe Journal of Parasitology vol 48 pp 715ndash718 1962

[33] J A Scott ldquoAn experimental study of the development of Ancy-lostoma caninum in normal and abnormal hostsrdquo AmericanJournal of Epidemiology vol 8 no 2 pp 158ndash204 1928

[34] J A Scott ldquoExperimental demonstration of a strain of the doghookworm Ancylostoma caninum especially adapted to thecatrdquo Journal for Parasitology vol 3 pp 209ndash215 1929

[35] O R Mccoy ldquoImmunity reactions of the dog against hook-worm (Ancylostoma caninum) under conditions of repeatedinfectionrdquo American Journal of Epidemiology vol 14 no 2 pp268ndash303 1931

[36] A O Foster and W W Cort ldquoThe effect of a deficient dieton the susceptibility of dogs and cats to non-specific strains ofhookwormsrdquo American Journal of Epidemiology vol 16 no 2pp 582ndash601 1932

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

Table 2 Previous investigations of hookworm species in cats worldwideCountry (area) Detection method Species Prevalence Source of animal Reference

Australia (Sydney) Necropsy A tubaeforme 35 Pound [6]U stenocephala 05 (119899 = 404)

Australia (Tasmania) Necropsy U stenocephala 2 (119899 = 86) Feral cats [18]Australia (Brsibane) Necropsy A tubaeforme 19 (119899 = 404) Pound [7]Australia (Brisbane) Necropsy A tubaeforme 81 Pound [19]Australia (Adelaide) Fecal exam U stenocephala 03 (119899 = 376) Vet practice [20]Australia (Kimberley) Necropsy A tubaeforme 20 (119899 = 34) Aboriginal community [21]Australia (Northern territory) Necropsy A tubaeforme 13 Feral cats [22]

Australia (countrywide) PCR-RFLP A tubaeforme 70 (119899 = 10) Refuge and pet cats [11]A caninum 30 (119899 = 10)

Brazil (Rio de Janeiro) Necropsy A braziliense 659 (119899 = 135) Cat (shelters and ZoonosesControl Center)

[23]A tubaeforme 89 (119899 = 135)

China (Sichuan) Necropsy

A caninum 25 (119899 = 20)

Domestic cat [15]A braziliense 10 (119899 = 20)A ceylanicum 5 (119899 = 20)U stenocephala 10 (119899 = 20)

China (Sichuan) NecropsyA caninum 51 (119899 = 149)

Domestic cat [16]A braziliense 174 (119899 = 149)U stenocephala 14 (119899 = 149)

Costa Rica (San Isidro ELGeneral) Fecal exam A tubaeforme 11 (119899 = 9) Refuge cat [24]

Thailand (Prachin Buri) A ceylanicumA caninum

9223 Cat [14]

Italy (central) Larvae (L3) exam A tubaeforme 12 (119899 = 81) Cat (pet) [25]U stenocephala 37 (119899 = 81)

Malaysia (West Malaysia) PCR-sequencing A ceylanicumA braziliense

261 (119899 = 23)43 (119899 = 23) Domestic cat [26]

Qatar (Doha) Necropsy A tubaeforme 147 (119899 = 658) Cat (feral) [27]Spain (mid-Ebro Valley) Necropsy A tubaeforme 293 (119899 = 58) Cat (stray) [28]

USA (Florida) Necropsy A tubaeforme 75 (119899 = 60) Cat (feral) [29]A braziliense 33 (119899 = 60)

USA (Pennsylvania) Fecal exam A tubaeforme 05 (119899 = 1566) Cat (pet) [30]

length presented by the A caninum ITS region (AM850105AM850106 EU159415 and EU159416) isolated from dogs inthis area DNA sequences were assembled using DNAStar(version 7 Madison WI USA) and multiple-sequence align-ment was performed with MegAlign program Comparativeanalysis of the three complete ITS sequences obtained fromcat-derived A caninum showed the same length (738 bp) ofdog-derived A caninum with a similarity of 992ndash997However compared to the ITS sequences of dog-derivedA caninum isolates (AM850105 AM850106 EU159415 andEU159416) the sequence variation range was 986ndash100where only one cat isolate (M63) showed 100 sequencesimilarity compared with two dog-derived A caninum iso-lates (AM850106 and EU159416) in the same studied areaThe sequence similarities of the cat-derived A caninum withA ceylanicum (KC755027) A tubaeforme (JQ812691) and Abraziliense (JQ812692) were 970ndash973 973ndash976 and916ndash921 respectively

4 Discussion

As early as the 19th century hookworms in cats and dogshad been described by Zedler (1800) and Ercolani (1859)[31] For nearly 100 years the common hookworm of bothdogs and cats was referred as Ancylostoma caninum [32]Later then some authors stated that it was difficult to infectdogs with larvae from cats and vice versa [32ndash36] Studiesin Europe Africa and Australia described themorphologicaldifferences of hookworms obtained from dogs and catswhere A caninumwas distinguished from A tubaeforme andwas thought to be host-specific for dogs [31 32]

To date feline hookworms (including A tubaeforme Abraziliense A ceylanicum A caninumand U stenocephala)had been reported nineteen times worldwide (Table 2) Obvi-ouslyA tubaeformewas themost common specieswhichwasreported in Australia (6 times) [6 7 11 19 21 22] USA (2times) [29 30] once in South America (Brazil) [23] Central

BioMed Research International 5

America (Costa Rica) [24] Europe (Italy Spain) [25 28] andMiddle East (Qatar) as well [27] while this parasite had notbeen reported fromcats inChina (EastAsia) [15 16]Malaysia(Southeast Asia) [26] andThailand [14]

In China A caninum was reported twice in Sichuanprovince southwest China with prevalence of 25 and 51respectively [15 16] while in our survey overall higher preva-lence (951) of A caninum infections in cats was detectedin Guangzhou (southern China) Our results suggested thatthe predominant species of hookworms in cats was Acaninum in China while A tubaeforme was considered tobe the predominant species in Australia [11] which stronglysupports our suggestion that the prevalent species is relatedto its geographical distribution as well as cats could be themain host for A caninum in China

The first cat-derived A caninum complete ITS sequences(GenBank KC755026 KC755028 and KC755029) are pre-sented in our study The length of obtained sequences(738 bp) was identical to the dog-derived A caninum ITSsequence as well as the sequence similarity range was 986ndash100 In addition A caninum from both cats and dogs wasconnected in a single cluster in the constructed phylogenetictree This finding indicated that there could be a cross-infection of A caninum between dogs and cats in the studiedareas

In conclusion the results of this study demonstratedthat The PCR-RFLP technique described in this study was arapid and straightforward method for the identification anddiscrimination of A caninum Moreover the ITS sequencescould be used to identify this hookworm species from differ-ent local hosts Current information regarding the prevalenceof A caninum showed possible cross-infections betweendifferent hosts Therefore it is imperative to have currentinformation regarding the prevalence of this hookwormand the associated risk factors of this infection This willallow a more effective implementation of strategic controlprogrammes for hookworm infections

Acknowledgments

This work was funded by the National Natural ScienceFoundation of China (Grant nos 30972179 and 31272551)The authors would like to thank Xu-hui Lin and the humaneshelterrsquo personnel for helping them in collecting all of thesamples

References

[1] L M C e Silva R R C Miranda H A Santos and E ML Rabelo ldquoDifferential diagnosis of dog hookworms based onPCR-RFLP from the ITS region of their rDNArdquo VeterinaryParasitology vol 140 no 3-4 pp 373ndash377 2006

[2] S Abubucker J Martin Y Yin et al ldquoThe canine hookwormgenome analysis and classification of Ancylostoma caninumsurvey sequencesrdquoMolecular and Biochemical Parasitology vol157 no 2 pp 187ndash192 2008

[3] M V Periago and J M Bethony ldquoHookworm virulence factorsmaking themost of the hostrdquoMicrobes and Infection vol 14 pp1451ndash1464 2012

[4] Y Yang J Zheng and J Chen ldquoCloning sequencing andphylogenetic analysis of the small GTPase gene cdc-42 fromAncylostoma caninumrdquo Experimental Parasitology vol 132 pp550ndash555 2012

[5] P Setasuban and A H Waddell ldquoHookworms in cats and dogsin Queenslandrdquo Australian Veterinary Journal vol 49 no 2 p110 1973

[6] J D Kelly and B K Y Ng ldquoHelminth parasites of dogs and catsI prevalence in urban environments in Australiardquo AustralianVeterinary Practitioner vol 5 pp 133ndash141 1975

[7] S L Wilson-Hanson and CW Prescott ldquoA survey for parasitesin catsrdquoAustralian Veterinary Journal vol 59 no 6 p 194 1982

[8] I Beveridge ldquoAustralian hookworms (Ancylostomatoidea) Areview of the species present their distributions and biogeo-graphical originsrdquo Parassitologia vol 44 no 1-2 pp 83ndash882002

[9] R B Gasser L E Stewart and R Speare ldquoGenetic markersin ribosomal DNA for hookworm identificationrdquo Acta Tropicavol 62 no 1 pp 15ndash21 1996

[10] P Prociv and J Croese ldquoHuman enteric infection with Ancy-lostoma caninum Hookworms reappraised in the light of aldquonewrdquo zoonosisrdquo Acta Tropica vol 62 no 1 pp 23ndash44 1996

[11] C S Palmer R J Traub I D Robertson et al ldquoThe veterinaryand public health significance of hookworm in dogs andcats in Australia and the status of A ceylanicumrdquo VeterinaryParasitology vol 145 no 3-4 pp 304ndash313 2007

[12] X Z Li R F Yan andXG Li ldquoInvestigation on gastrointestinalparasites of dogs inNanjingrdquoAnimal Husbandry andVeterinaryMedicine vol 43 pp 72ndash73 2011 (Chinese)

[13] J G Sun ldquoInvestigation on parasites of domestic dogs and otherlivestock in Cangxi County Sichuan Provincerdquo Chinese Journalof Zoonoses vol 3 article 53 1987 (Chinese)

[14] P Setasuban S Vajrasthira and C Muennoo ldquoPrevalenceand zoonotic potential of Ancylostoma ceylanicum in cats inThailandrdquo Southeast Asian Journal of Tropical Medicine andPublic Health vol 7 no 4 pp 534ndash539 1976

[15] M F Yang ldquoInvestigation of felid parasite in Sichuanrdquo SichuanJournal of Agricultural Science vol 2 pp 45ndash48 1987 (Chinese)

[16] Y Feng Q He and J P Li ldquoInvestigation of canine and felidparasite in TongjiangCountyrdquoModernAgricultural Sciences andTechnology vol 11 pp 354ndash355 2011 (Chinese)

[17] J DThompson T J Gibson F Plewniak F Jeanmougin andDG Higgins ldquoTheClustal X windows interface flexible strategiesformultiple sequence alignment aided by quality analysis toolsrdquoNucleic Acids Research vol 24 pp 4876ndash4882 1997

[18] G G Gregory and B L Munday ldquoInternal parasites of feralcats from the TasmanianMidlands and King IslandrdquoAustralianVeterinary Journal vol 52 no 7 pp 317ndash320 1976

[19] C W Prescott Parasitic Diseases of the Cat in Australia TheUniversity of Sydney Sydney Australia 1984

[20] E Moore and M G OrsquoCallaghan ldquoHelminths of dogs andcats determined by faecal examinations in Adelaide SouthAustraliardquo Australian Veterinary Journal vol 62 no 6 pp 198ndash199 1985

[21] B P Meloni R C AThompson R M Hopkins J A Reynold-son and M Gracey ldquoThe prevalence of Giardia and otherintestinal parasites in children dogs and cats from Aboriginalcommunities in the Kimberleyrdquo Medical Journal of Australiavol 158 no 3 pp 157ndash159 1993

[22] MGOrsquoCallaghan and I Beveridge ldquoGastro-intestinal parasitesof feral cats in the Northern Territoryrdquo Transactions of the RoyalSociety of South Australia vol 120 no 3-4 pp 175ndash176 1996

6 BioMed Research International

[23] N Labarthe M L Serrao A M R Ferreira N K O Almeidaand J Guerrero ldquoA survey of gastrointestinal helminths in catsof the metropolitan region of Rio de Janeiro Brazilrdquo VeterinaryParasitology vol 123 no 1-2 pp 133ndash139 2004

[24] AV Scorza CDuncan LMiles andMR Lappin ldquoPrevalenceof selected zoonotic and vector-borne agents in dogs and catsin Costa RicardquoVeterinary Parasitology vol 183 no 1-2 pp 178ndash183 2011

[25] F Riggio R Mannella G Ariti and S Perrucci ldquoIntestinaland lung parasites in owned dogs and cats from central ItalyrdquoVeterinary Parasitology vol 193 pp 78ndash84 2013

[26] R Ngui Y A L Lim R Traub R Mahmud and M SMistam ldquoEpidemiological and genetic data supporting thetransmission of Ancylostoma ceylanicum among human anddomestic animalsrdquo PLoS Neglected Tropical Diseases vol 6 no2 Article ID e1522 2012

[27] M A Abu-Madi J M Behnke K S Prabhaker R Al-Ibrahimand J W Lewis ldquoIntestinal helminths of feral cat populationsfrom urban and suburban districts of Qatarrdquo Veterinary Para-sitology vol 168 no 3-4 pp 284ndash292 2010

[28] C Calvete J Lucientes J A Castillo et al ldquoGastrointestinalhelminth parasites in stray cats from the mid-Ebro ValleySpainrdquo Veterinary Parasitology vol 75 no 2-3 pp 235ndash2401998

[29] T C Anderson G W Foster and D J Forrester ldquoHookwormsof feral cats in Floridardquo Veterinary Parasitology vol 115 no 1pp 19ndash24 2003

[30] M C Gates and T J Nolan ldquoEndoparasite prevalence andrecurrence across different age groups of dogs and catsrdquoVeterinary Parasitology vol 166 no 1-2 pp 153ndash158 2009

[31] E Biocca ldquoRediscrizione di Ancylostoma tubaeforme (Zedler1800) parassita del gatto considerate erroneamente sinonimodi Ancylostoma caninum (Ercolani 1859) parassita del camerdquoRivista di Parassitologia vol 15 pp 267ndash278 1954

[32] R B Burrows ldquoComparative morphology of Ancylostomatubaeforme (Zeder 1800) and Ancylostoma caninum (Ercolani1859)rdquoThe Journal of Parasitology vol 48 pp 715ndash718 1962

[33] J A Scott ldquoAn experimental study of the development of Ancy-lostoma caninum in normal and abnormal hostsrdquo AmericanJournal of Epidemiology vol 8 no 2 pp 158ndash204 1928

[34] J A Scott ldquoExperimental demonstration of a strain of the doghookworm Ancylostoma caninum especially adapted to thecatrdquo Journal for Parasitology vol 3 pp 209ndash215 1929

[35] O R Mccoy ldquoImmunity reactions of the dog against hook-worm (Ancylostoma caninum) under conditions of repeatedinfectionrdquo American Journal of Epidemiology vol 14 no 2 pp268ndash303 1931

[36] A O Foster and W W Cort ldquoThe effect of a deficient dieton the susceptibility of dogs and cats to non-specific strains ofhookwormsrdquo American Journal of Epidemiology vol 16 no 2pp 582ndash601 1932

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

America (Costa Rica) [24] Europe (Italy Spain) [25 28] andMiddle East (Qatar) as well [27] while this parasite had notbeen reported fromcats inChina (EastAsia) [15 16]Malaysia(Southeast Asia) [26] andThailand [14]

In China A caninum was reported twice in Sichuanprovince southwest China with prevalence of 25 and 51respectively [15 16] while in our survey overall higher preva-lence (951) of A caninum infections in cats was detectedin Guangzhou (southern China) Our results suggested thatthe predominant species of hookworms in cats was Acaninum in China while A tubaeforme was considered tobe the predominant species in Australia [11] which stronglysupports our suggestion that the prevalent species is relatedto its geographical distribution as well as cats could be themain host for A caninum in China

The first cat-derived A caninum complete ITS sequences(GenBank KC755026 KC755028 and KC755029) are pre-sented in our study The length of obtained sequences(738 bp) was identical to the dog-derived A caninum ITSsequence as well as the sequence similarity range was 986ndash100 In addition A caninum from both cats and dogs wasconnected in a single cluster in the constructed phylogenetictree This finding indicated that there could be a cross-infection of A caninum between dogs and cats in the studiedareas

In conclusion the results of this study demonstratedthat The PCR-RFLP technique described in this study was arapid and straightforward method for the identification anddiscrimination of A caninum Moreover the ITS sequencescould be used to identify this hookworm species from differ-ent local hosts Current information regarding the prevalenceof A caninum showed possible cross-infections betweendifferent hosts Therefore it is imperative to have currentinformation regarding the prevalence of this hookwormand the associated risk factors of this infection This willallow a more effective implementation of strategic controlprogrammes for hookworm infections

Acknowledgments

This work was funded by the National Natural ScienceFoundation of China (Grant nos 30972179 and 31272551)The authors would like to thank Xu-hui Lin and the humaneshelterrsquo personnel for helping them in collecting all of thesamples

References

[1] L M C e Silva R R C Miranda H A Santos and E ML Rabelo ldquoDifferential diagnosis of dog hookworms based onPCR-RFLP from the ITS region of their rDNArdquo VeterinaryParasitology vol 140 no 3-4 pp 373ndash377 2006

[2] S Abubucker J Martin Y Yin et al ldquoThe canine hookwormgenome analysis and classification of Ancylostoma caninumsurvey sequencesrdquoMolecular and Biochemical Parasitology vol157 no 2 pp 187ndash192 2008

[3] M V Periago and J M Bethony ldquoHookworm virulence factorsmaking themost of the hostrdquoMicrobes and Infection vol 14 pp1451ndash1464 2012

[4] Y Yang J Zheng and J Chen ldquoCloning sequencing andphylogenetic analysis of the small GTPase gene cdc-42 fromAncylostoma caninumrdquo Experimental Parasitology vol 132 pp550ndash555 2012

[5] P Setasuban and A H Waddell ldquoHookworms in cats and dogsin Queenslandrdquo Australian Veterinary Journal vol 49 no 2 p110 1973

[6] J D Kelly and B K Y Ng ldquoHelminth parasites of dogs and catsI prevalence in urban environments in Australiardquo AustralianVeterinary Practitioner vol 5 pp 133ndash141 1975

[7] S L Wilson-Hanson and CW Prescott ldquoA survey for parasitesin catsrdquoAustralian Veterinary Journal vol 59 no 6 p 194 1982

[8] I Beveridge ldquoAustralian hookworms (Ancylostomatoidea) Areview of the species present their distributions and biogeo-graphical originsrdquo Parassitologia vol 44 no 1-2 pp 83ndash882002

[9] R B Gasser L E Stewart and R Speare ldquoGenetic markersin ribosomal DNA for hookworm identificationrdquo Acta Tropicavol 62 no 1 pp 15ndash21 1996

[10] P Prociv and J Croese ldquoHuman enteric infection with Ancy-lostoma caninum Hookworms reappraised in the light of aldquonewrdquo zoonosisrdquo Acta Tropica vol 62 no 1 pp 23ndash44 1996

[11] C S Palmer R J Traub I D Robertson et al ldquoThe veterinaryand public health significance of hookworm in dogs andcats in Australia and the status of A ceylanicumrdquo VeterinaryParasitology vol 145 no 3-4 pp 304ndash313 2007

[12] X Z Li R F Yan andXG Li ldquoInvestigation on gastrointestinalparasites of dogs inNanjingrdquoAnimal Husbandry andVeterinaryMedicine vol 43 pp 72ndash73 2011 (Chinese)

[13] J G Sun ldquoInvestigation on parasites of domestic dogs and otherlivestock in Cangxi County Sichuan Provincerdquo Chinese Journalof Zoonoses vol 3 article 53 1987 (Chinese)

[14] P Setasuban S Vajrasthira and C Muennoo ldquoPrevalenceand zoonotic potential of Ancylostoma ceylanicum in cats inThailandrdquo Southeast Asian Journal of Tropical Medicine andPublic Health vol 7 no 4 pp 534ndash539 1976

[15] M F Yang ldquoInvestigation of felid parasite in Sichuanrdquo SichuanJournal of Agricultural Science vol 2 pp 45ndash48 1987 (Chinese)

[16] Y Feng Q He and J P Li ldquoInvestigation of canine and felidparasite in TongjiangCountyrdquoModernAgricultural Sciences andTechnology vol 11 pp 354ndash355 2011 (Chinese)

[17] J DThompson T J Gibson F Plewniak F Jeanmougin andDG Higgins ldquoTheClustal X windows interface flexible strategiesformultiple sequence alignment aided by quality analysis toolsrdquoNucleic Acids Research vol 24 pp 4876ndash4882 1997

[18] G G Gregory and B L Munday ldquoInternal parasites of feralcats from the TasmanianMidlands and King IslandrdquoAustralianVeterinary Journal vol 52 no 7 pp 317ndash320 1976

[19] C W Prescott Parasitic Diseases of the Cat in Australia TheUniversity of Sydney Sydney Australia 1984

[20] E Moore and M G OrsquoCallaghan ldquoHelminths of dogs andcats determined by faecal examinations in Adelaide SouthAustraliardquo Australian Veterinary Journal vol 62 no 6 pp 198ndash199 1985

[21] B P Meloni R C AThompson R M Hopkins J A Reynold-son and M Gracey ldquoThe prevalence of Giardia and otherintestinal parasites in children dogs and cats from Aboriginalcommunities in the Kimberleyrdquo Medical Journal of Australiavol 158 no 3 pp 157ndash159 1993

[22] MGOrsquoCallaghan and I Beveridge ldquoGastro-intestinal parasitesof feral cats in the Northern Territoryrdquo Transactions of the RoyalSociety of South Australia vol 120 no 3-4 pp 175ndash176 1996

6 BioMed Research International

[23] N Labarthe M L Serrao A M R Ferreira N K O Almeidaand J Guerrero ldquoA survey of gastrointestinal helminths in catsof the metropolitan region of Rio de Janeiro Brazilrdquo VeterinaryParasitology vol 123 no 1-2 pp 133ndash139 2004

[24] AV Scorza CDuncan LMiles andMR Lappin ldquoPrevalenceof selected zoonotic and vector-borne agents in dogs and catsin Costa RicardquoVeterinary Parasitology vol 183 no 1-2 pp 178ndash183 2011

[25] F Riggio R Mannella G Ariti and S Perrucci ldquoIntestinaland lung parasites in owned dogs and cats from central ItalyrdquoVeterinary Parasitology vol 193 pp 78ndash84 2013

[26] R Ngui Y A L Lim R Traub R Mahmud and M SMistam ldquoEpidemiological and genetic data supporting thetransmission of Ancylostoma ceylanicum among human anddomestic animalsrdquo PLoS Neglected Tropical Diseases vol 6 no2 Article ID e1522 2012

[27] M A Abu-Madi J M Behnke K S Prabhaker R Al-Ibrahimand J W Lewis ldquoIntestinal helminths of feral cat populationsfrom urban and suburban districts of Qatarrdquo Veterinary Para-sitology vol 168 no 3-4 pp 284ndash292 2010

[28] C Calvete J Lucientes J A Castillo et al ldquoGastrointestinalhelminth parasites in stray cats from the mid-Ebro ValleySpainrdquo Veterinary Parasitology vol 75 no 2-3 pp 235ndash2401998

[29] T C Anderson G W Foster and D J Forrester ldquoHookwormsof feral cats in Floridardquo Veterinary Parasitology vol 115 no 1pp 19ndash24 2003

[30] M C Gates and T J Nolan ldquoEndoparasite prevalence andrecurrence across different age groups of dogs and catsrdquoVeterinary Parasitology vol 166 no 1-2 pp 153ndash158 2009

[31] E Biocca ldquoRediscrizione di Ancylostoma tubaeforme (Zedler1800) parassita del gatto considerate erroneamente sinonimodi Ancylostoma caninum (Ercolani 1859) parassita del camerdquoRivista di Parassitologia vol 15 pp 267ndash278 1954

[32] R B Burrows ldquoComparative morphology of Ancylostomatubaeforme (Zeder 1800) and Ancylostoma caninum (Ercolani1859)rdquoThe Journal of Parasitology vol 48 pp 715ndash718 1962

[33] J A Scott ldquoAn experimental study of the development of Ancy-lostoma caninum in normal and abnormal hostsrdquo AmericanJournal of Epidemiology vol 8 no 2 pp 158ndash204 1928

[34] J A Scott ldquoExperimental demonstration of a strain of the doghookworm Ancylostoma caninum especially adapted to thecatrdquo Journal for Parasitology vol 3 pp 209ndash215 1929

[35] O R Mccoy ldquoImmunity reactions of the dog against hook-worm (Ancylostoma caninum) under conditions of repeatedinfectionrdquo American Journal of Epidemiology vol 14 no 2 pp268ndash303 1931

[36] A O Foster and W W Cort ldquoThe effect of a deficient dieton the susceptibility of dogs and cats to non-specific strains ofhookwormsrdquo American Journal of Epidemiology vol 16 no 2pp 582ndash601 1932

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

[23] N Labarthe M L Serrao A M R Ferreira N K O Almeidaand J Guerrero ldquoA survey of gastrointestinal helminths in catsof the metropolitan region of Rio de Janeiro Brazilrdquo VeterinaryParasitology vol 123 no 1-2 pp 133ndash139 2004

[24] AV Scorza CDuncan LMiles andMR Lappin ldquoPrevalenceof selected zoonotic and vector-borne agents in dogs and catsin Costa RicardquoVeterinary Parasitology vol 183 no 1-2 pp 178ndash183 2011

[25] F Riggio R Mannella G Ariti and S Perrucci ldquoIntestinaland lung parasites in owned dogs and cats from central ItalyrdquoVeterinary Parasitology vol 193 pp 78ndash84 2013

[26] R Ngui Y A L Lim R Traub R Mahmud and M SMistam ldquoEpidemiological and genetic data supporting thetransmission of Ancylostoma ceylanicum among human anddomestic animalsrdquo PLoS Neglected Tropical Diseases vol 6 no2 Article ID e1522 2012

[27] M A Abu-Madi J M Behnke K S Prabhaker R Al-Ibrahimand J W Lewis ldquoIntestinal helminths of feral cat populationsfrom urban and suburban districts of Qatarrdquo Veterinary Para-sitology vol 168 no 3-4 pp 284ndash292 2010

[28] C Calvete J Lucientes J A Castillo et al ldquoGastrointestinalhelminth parasites in stray cats from the mid-Ebro ValleySpainrdquo Veterinary Parasitology vol 75 no 2-3 pp 235ndash2401998

[29] T C Anderson G W Foster and D J Forrester ldquoHookwormsof feral cats in Floridardquo Veterinary Parasitology vol 115 no 1pp 19ndash24 2003

[30] M C Gates and T J Nolan ldquoEndoparasite prevalence andrecurrence across different age groups of dogs and catsrdquoVeterinary Parasitology vol 166 no 1-2 pp 153ndash158 2009

[31] E Biocca ldquoRediscrizione di Ancylostoma tubaeforme (Zedler1800) parassita del gatto considerate erroneamente sinonimodi Ancylostoma caninum (Ercolani 1859) parassita del camerdquoRivista di Parassitologia vol 15 pp 267ndash278 1954

[32] R B Burrows ldquoComparative morphology of Ancylostomatubaeforme (Zeder 1800) and Ancylostoma caninum (Ercolani1859)rdquoThe Journal of Parasitology vol 48 pp 715ndash718 1962

[33] J A Scott ldquoAn experimental study of the development of Ancy-lostoma caninum in normal and abnormal hostsrdquo AmericanJournal of Epidemiology vol 8 no 2 pp 158ndash204 1928

[34] J A Scott ldquoExperimental demonstration of a strain of the doghookworm Ancylostoma caninum especially adapted to thecatrdquo Journal for Parasitology vol 3 pp 209ndash215 1929

[35] O R Mccoy ldquoImmunity reactions of the dog against hook-worm (Ancylostoma caninum) under conditions of repeatedinfectionrdquo American Journal of Epidemiology vol 14 no 2 pp268ndash303 1931

[36] A O Foster and W W Cort ldquoThe effect of a deficient dieton the susceptibility of dogs and cats to non-specific strains ofhookwormsrdquo American Journal of Epidemiology vol 16 no 2pp 582ndash601 1932

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology