Veterinary Parasitology 172 (2010) 132–134

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Veterinary Parasitology

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Short communication

Identification of Cryptosporidium xiaoi in diarrhoeic goat kids(Capra hircus) in Spain

Pablo Díaza,∗, Joaquín Quílezb, Guy Robinsonc, Rachel M. Chalmersc,Pablo Díez-Banosa, Patrocinio Morrondoa

a Department of Animal Pathology, Faculty of Veterinary Sciences, University of Santiago de Compostela, 27002 Lugo, Spainb Department of Animal Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spainc UK Cryptosporidium Reference Unit, NPHS Microbiology Swansea, Singleton Hospital, Swansea SA2 8QA, UK

a r t i c l e i n f o

Article history:Received 12 February 2010Received in revised form 19 April 2010Accepted 26 April 2010

a b s t r a c t

Faecal specimens from five diarrhoeic goat kids (Capra hircus) younger than 21 days werecollected in a goat farm in Galicia (NW Spain) and examined for the presence of Cryp-tosporidium oocysts. Two Cryptosporidium-positive isolates were detected and selected formolecular examination. A banding pattern indicative of Cryptosporidium bovis was obtained

Keywords:Cryptosporidium xiaoiGoat kidsNeonatal diarrhoea

after restriction analyses of PCR products from small-subunit rRNA genes. However, bothpositive isolates were identified as Cryptosporidium xiaoi by sequence analyses of SSU rRNAand actin genes. Our results demonstrate that domestic goats are a host for C. xiaoi andsuggest that this parasite species may be involved in the aetiology of neonatal diarrhoeic

farms.

Spain outbreaks in goatSpain.

1. Introduction

Studies of the prevalence of Cryptosporidium in farmanimals have revealed that ruminants are the primary non-human species impacted by cryptosporidiosis, althoughmost data are related to cattle. However, cryptosporidio-sis is also a well-recognized cause of neonatal diarrhoeain domestic small ruminants, including sheep and goats,which represent an important sector of the agriculturaleconomy in many countries. Cryptosporidial infection ingoat kids was first described in Australia and then asso-

ciated with diarrhoea and high mortality in kids aged1–2 weeks in different parts of the world, with preva-lence rates ranging from <5% to >35% (de Graaf et al.,1999; Robertson, 2009). Nevertheless, the Cryptosporidium

∗ Corresponding author at: Parasitología y Enfermedades parasitarias,Departamento de Patología Animal, Facultad de Veterinaria, Campus Uni-versitario s/n, 27002 Lugo, Spain. Tel.: +34 982252350x22129;fax: +34 982252195.

E-mail address: pablo.diaz@usc.es (P. Díaz).

0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.vetpar.2010.04.029

This is the first published description of C. xiaoi in goat kids in

© 2010 Elsevier B.V. All rights reserved.

species/genotypes infecting goats are not well docu-mented and its impact as a source for human infection iscontroversial, mainly because molecular studies of Cryp-tosporidium isolates from goats are limited and restricted tomodest numbers of specimens. The zoonotic species Cryp-tosporidium parvum has been identified in diarrhoeic andasymptomatic goat kids in Australia, Cyprus, Zambia andBelgium (Morgan et al., 1998; Goma et al., 2007; Geurdenet al., 2008) as well as adult goats from the United King-dom, Italy and the Czech Republic (Chalmers et al., 2002;Wu et al., 2003; Hajdusek et al., 2004, O’Brien et al., 2008).Additionally, Cryptosporidium hominis and Cryptosporid-ium bovis-like genotype were described in one adult goateach in the United Kingdom (Giles et al., 2009) and China(Karanis et al., 2007), respectively. The latter genotype hasrecently been named as the new species Cryptosporidiumxiaoi, based on genetic differences at three independent loci

(Fayer and Santín, 2009).

Spain has a sizable goat population, with around 36,000herds and 2.5 million animals in 2008 (Ministry of Environ-ment, and Rural and Marine Affairs, http://www.marm.es).Cryptosporidium has been reported as the most frequently

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solated pathogen in goat kids with gastrointestinal dis-ase in some areas of the country (Matos-Fernández et al.,994; Munoz-Fernández et al., 1996). Herd-level preva-

ence rates range from 40% to 100% and 10% to 70% ofnfected diarrhoeic kids, but the geographic distribution ofryptosporidium species in this ruminant species remainsnclear (Matos-Fernández et al., 1994; Munoz-Fernándezt al., 1996; Sanz Ceballos et al., 2009). The only molec-lar study reported so far in goat herds in Spain wasonducted in the northeast of the country, revealing theresence of only C. parvum in 17 diarrhoeic goat kids fromfarms (Quílez et al., 2008). Samples analysed in the cur-

ent work were part of a larger study (Díaz et al., in press) tonvestigate the occurrence and zoonotic potential of Cryp-osporidium isolates from pre-weaned domestic ruminantsn Galicia, an important livestock-rearing area located inorthwestern Spain.

. Materials and methods

Faecal specimens from five diarrhoeic goat kids (Capraircus) younger than 21 days were collected using cot-on rectal swabs in a Murciano-Granadina goat farm inalicia (NW Spain). Samples were examined for the pres-nce of Cryptosporidium oocysts by microscopy of carboluchsin negative-stained smears (Heine, 1982), and posi-ive specimens were selected for molecular examination.ocyst recovery and DNA extraction were performed asescribed previously (Quílez et al., 2008). Cryptosporidiumpecies were determined by a nested PCR of a small-subunitSSU) rRNA gene fragment and restriction fragment lengtholymorphism (RFLP) analysis with the endonucleases SspInd VspI using the primers described previously (Jiang etl., 2005). Positive isolates were further characterized bynested PCR targeting the actin gene (∼830 bp) (Ng et al.,006). Non-acetylated BSA (0.5 mg/ml) was added to theCR master mixes to reduce any potential inhibition in theample (Jiang et al., 2005). Secondary PCR products of SSURNA and actin genes were sequenced in order to confirmhe identity of both positive isolates.

. Results and discussion

Two goat kids tested positive for Cryptosporidium byicroscopy after negative staining with carbol fuschin.restriction pattern indicative of C. bovis was obtained

fter digestion with the endonucleases SspI and VspI (Fengt al., 2007). However, DNA sequencing of the SSU rRNAene of both positive isolates yielded partial sequencesith a 99.88% (812/813 bp) similarity to C. xiaoi. Specifi-

ally, our isolates showed a single nucleotide transversionn comparison to different C. xiaoi isolates from sheepn Norway, China and the United States (accession num-ers GQ337963, GU014552, FJ896051), and two nucleotideutations when compared to the C. xiaoi GU174541 fromgoat in China (Fayer and Santín, 2009; Wang et al., 2010;

obertson et al., in press; Liu et al., in press).

Sequence analysis of the actin gene revealed 99.88%852/853 bp) similarity with C. xiaoi isolates from sheepn Norway (GQ337964) and the United States (FJ896042),xcept for a G to A nucleotide transition at position 671

ogy 172 (2010) 132–134 133

(Fayer and Santín, 2009; Robertson et al., in press). Bycontrast, our isolates showed an inter-isolate variance of18 bp when compared with C. bovis AY741307 (Fayer etal., 2005). Representative nucleotide sequences have beendeposited in GenBank under accession numbers GU553016and GU553017.

C. xiaoi was described as a C. bovis-like genotype in agoat (Karanis et al., 2007) and a yak (Feng et al., 2007) inChina, but most natural infections have been reported insheep, probably as a consequence of the larger numbersof isolates analysed (Fayer and Santín, 2009). It is worthmentioning that no signs of disease were seen in these pre-vious reports, except for a C. bovis-like infected 8-day-oldlamb from Spain that died with acute diarrhoea and rapidweight loss, although the authors remarked that they hadnot proved that the clinical symptoms were really causedby the protozoan (Navarro-i-Martínez et al., 2007). It isalso interesting to note that attempts to experimentallyinfect three 36-week-old goat kids with a C. xiaoi isolatewere unsuccessful (Fayer and Santín, 2009). Future stud-ies should examine the potential for age-related C. xiaoiinfections in ovine and caprine hosts, as infections of theclosely related C. bovis in cattle appear to be age-related.Santín et al. (2004) reported that C. bovis was only foundin cattle up to the age of 7 months and so if C. xiaoi has asimilar host age preference it may explain the unsuccess-ful attempts at infecting 8–9-month-old goats (Fayer andSantín, 2009). Our results demonstrate that domestic goatsare a host for C. xiaoi and suggest that this parasite speciesmay be involved in the aetiology of neonatal diarrhoeic out-breaks in goat farms, bearing in mind the enteropathogenicrole of the protozoan. Nevertheless, this clinical syndromewas not necessarily attributed only to cryptosporidiosis,since neither bacterial nor viral infections were checked.This is the first published description of C. xiaoi in goat kidsin Spain.

Acknowledgements

This work was supported by a postdoctoral researchgrant (Xunta de Galicia, Spain) to P. Díaz and funds froma Spanish research program (AGL2009-10590). We wouldlike to thank Samuel Pereira for his help with collectingthe samples and UK Cryptosporidium Reference Unit staffStephen Hadfield and Kristin Elwin for scientific support.

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