Cryptosporidium cuniculus - new records in human and kangaroo in Australia
© Koehler et al.; licensee BioMed Central Ltd. 2014
Received: 2 September 2014
Accepted: 20 October 2014
Published: 30 October 2014
To date, Cryptosporidium cuniculus has been found exclusively in rabbits and humans. The present study provides the first published molecular evidence for C. cuniculus in an Australian human patient as well as a kangaroo.
Using PCR-based sequencing of regions in the actin, 60 kDa glycoprotein (gp60) and small subunit of ribosomal RNA (SSU) genes, we identified a new and unique C. cuniculus genotype (akin to VbA25) from a human, and C. cuniculus genotype VbA26 from an Eastern grey kangaroo (Macropus giganteus) in Australia.
The characterisation of these genotypes raises questions as to their potential to infect humans and/or other animals in Australia, given that C. cuniculus has been reported to cause cryptosporidiosis outbreaks in Europe.
KeywordsCryptosporidium cuniculus Australia Human Kangaroo Novel genotype
Cryptosporidium is a genus of apicomplexan protozoans that infect humans and other animal hosts, and frequently cause enteritis and associated diarrhoea . Most infections of humans are attributed to C. hominis and C. parvum, but C. canis, C. felis and C. meleagridis, which usually infect dogs, cats and birds, respectively, are also zoonotic . Interestingly, C. cuniculus, usually a parasite of rabbits, is genetically closely related to C. hominis and C. parvum, and has been responsible for outbreaks of cryptosporidiosis in humans in the UK -. In the first recorded outbreak, the infection source was water from a tank contaminated by C. cuniculus-infected European rabbit (Oryctolagus cuniculus), and the protist was identified with the aid of molecular tools .
Using the sequences of small subunit of ribosomal RNA (SSU), actin and/or Cryptosporidium oocyst wall protein (COWP) genes, C. cuniculus can be distinguished genetically from the closely related species C. hominis. In addition, C. cuniculus can be readily identified based on the sequence of the 60 kDa glycoprotein (gp60) gene. Using this locus, two distinct clades, designated Va and Vb, can be classified according to the number of TCA (serine)-tandem repeats in the microsatellite region (e.g., VaA26 = clade Va, with 26 TCA repeats) . Although there is no evidence to suggest that either clade is strictly host-specific, most cases described in humans relate to clade Va .
Since the first UK outbreak , there has been increasing interest in C. cuniculus due to the zoonotic threat that it poses , and this pathogen has been identified in humans and/or rabbits mainly in the UK and China ,-, with some reports from other countries including the Czech Republic, France and Nigeria -. In addition, a recent outbreak was associated with the deaths of 300 rabbits in Poland . Although C. cuniculus has been recorded in rabbits in south-eastern Australia ,, there is no published report of this pathogen from humans or other host species in Australia. Here, we report C. cuniculus genotypes originating from an Australian human patient and, for the first time, from an Eastern grey kangaroo (Macropus giganteus).
A faecal sample (collected in 2009) from a human known to be clinically affected by cryptosporidiosis was provided to us (anonymously) by the Microbiology Diagnostic Unit of the Public Health Laboratory (MDU/PHL) of the University of Melbourne. Faecal DNA was isolated following treatment with 10% polyvinyl-polypyrrolidone, using the standard QIAamp DNA Mini Kit (Qiagen, Germany) protocol. In addition, a faecal sample from M. giganteus was collected during a surveillance campaign of waterborne pathogens in wildlife within the drinking water catchment areas of Melbourne Water (www.melbournewater.com.au) in Victoria, Australia . This faecal sample was collected in August 2013 in the Yan Yean catchment (-37.619704; 144.901997) ; the species of kangaroo host was initially established by sight, according to Triggs , and then confirmed using an established PCR-based method (unpublished) for the sequencing of a mitochondrial cytochrome oxidase b gene region of M. giganteus from faecal DNA isolated using a kit (PowerSoil, Mo-Bio, California). Subsequently, regions of the actin, gp60 (two regions) and/or SSU genes of Cryptosporidium were amplified individually from each of the two faecal DNA samples by PCR and then sequenced as described previously . Nucleotide sequences were deposited in the GenBank database [GenBank: KM366138-KM366142].
C. cuniculus was also identified in the faecal sample from a M. giganteus individual from the Yan Yean catchment based on the SSU [GenBank: KM366142] (557 bp) and gp60 [GenBank: KM366140] (282 bp) sequences from amplicons produced. The gp60 sequence (VbA26) was similar to that from C. cuniculus (VbA25; with an additional serine repeat) [GenBank: KC283003] from the same catchment (2011), and identical to sequences [GenBank: HM852433 and KC283004] originating from C. cuniculus from the same lagomorph species from another Melbourne Water catchment (Sugar Loaf; -37.681999; 145.293841) in 2010.
To date, C. cuniculus has been found exclusively in rabbits and humans ,,. Here, we provide molecular evidence for a new C. cuniculus genotype in a human in Australia and, for the first time, for the occurrence of C. cuniculus in a kangaroo. In Victoria, Australia, as part of an ongoing pathogen detection program, in which we have molecularly tested >1500 faecal samples from kangaroos and wallabies (; Koehler et al. unpublished data), this is the first instance of C. cuniculus being detected in the faeces from any macropodid host. The presence of C. cuniculus DNA in the faecal sample from the M. giganteus individual indicates, but does not prove, that this macropod was infected with this pathogen. There is a remote possibility that C. cuniculus oocysts passed in faeces related to pseudoparasitism , as a result of the kangaroo ingesting such oocysts while grazing on grass contaminated by faeces from infected rabbits. Nonetheless, given that many kangaroos graze in some of Melbourne’s water catchment areas, where rabbits are common, we would have expected to identify C. cuniculus much more frequently by molecular testing than one of >1500 samples tested to date. Therefore, the probability that this M. giganteus individual harboured a cryptosporidial infection is considered high.
The zoonotic risk of C. cuniculus to human health is clearly evidenced by the first outbreak in the UK in 2008, in which 29 people reported illness after drinking water contaminated with C. cuniculus from a rabbit . In addition, recently, the dramatic effect of C. cuniculus on its lagomorph host was demonstrated when 300 rabbits were reported to have died from cryptosporidiosis linked to this species of Cryptosporidium (c.f. ). The occurrence of C. cuniculus genotypes in rabbits  and, here, in M. giganteus suggests that such genotypes might be able to spread to other native mammals and/or humans in Australia. Therefore, there is a need to diligently monitor Cryptosporidium in the vicinity of drinking water catchments (c.f. ) and in drinking water. The need for monitoring by molecular tools is particularly crucial in Victoria and other states of Australia, where rabbit populations have flourished ever since their introduction in 1859, causing a widespread degradation of natural ecosystems . Such monitoring is also critical, given that people in Victoria consume unfiltered drinking water from natural catchment areas, and that currently used water treatment processes/procedures do not destroy oocysts of Cryptosporidium.
Sequencing of regions in the actin, gp60 and SSU genes has led to the identification of a new and unique C. cuniculus genotype (similar to VbA25 from a human); for the first time, C. cuniculus (genotype VbA26) has been found in an Eastern grey kangaroo (M. giganteus) in Australia. The characterisation of this and similar genotypes raises questions as to their potential to infect humans and/or other animals in Australia, given that C. cuniculus has been reported to cause cryptosporidiosis outbreaks in Europe.
AK carried out the molecular work and drafted the manuscript. MW supplied the samples. SH participated in the coordination of the study. RG participated in the design, analysis and drafting of the manuscript. All authors read and approved the final version of the manuscript.
Funds from the Australian Research Council (ARC), the National Health and Medical Research Council (NHMRC) and Melbourne Water Corporation are gratefully acknowledged.
- Xiao L, Fayer R: Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission. Int J Parasitol. 2008, 38: 1239-1255. 10.1016/j.ijpara.2008.03.006.View ArticlePubMedGoogle Scholar
- Xiao L, Feng Y: Zoonotic cryptosporidiosis. FEMS Immunol Med Microbiol. 2008, 52: 309-323. 10.1111/j.1574-695X.2008.00377.x.View ArticlePubMedGoogle Scholar
- Chalmers RM, Robinson G, Elwin K, Hadfield SJ, Xiao L, Ryan UM, Modha D, Mallaghan C:Cryptosporidium rabbit genotype, a newly identified human pathogen. Emerg Infect Dis. 2009, 15: 829-830. 10.3201/eid1505.081419.PubMed CentralView ArticlePubMedGoogle Scholar
- Robinson G, Wright S, Elwin K, Hadfield SJ, Katzer F, Bartley PM, Hunter PR, Nath M, Innes EA, Chalmers RM: Re-description of Cryptosporidium cuniculus Inman and Takeuchi, 1979 (Apicomplexa: Cryptosporidiidae): morphology, biology and phylogeny. Int J Parasitol. 2010, 40: 1539-1548. 10.1016/j.ijpara.2010.05.010.View ArticlePubMedGoogle Scholar
- Puleston RL, Mallaghan CM, Modha DE, Hunter PR, Nguyen-Van-Tam JS, Regan CM, Nichols GL, Chalmers RM: The first recorded outbreak of cryptosporidiosis due to Cryptosporidium cuniculus (formerly rabbit genotype), following a water quality incident. J Water Health. 2014, 12: 41-50. 10.2166/wh.2013.097.View ArticlePubMedGoogle Scholar
- Robinson G, Chalmers R: The European rabbit (Oryctolagus cuniculus), a source of zoonotic cryptosporidiosis. Zoonoses Public Health. 2010, 57: e1-e13. 10.1111/j.1863-2378.2009.01308.x.View ArticlePubMedGoogle Scholar
- Zhang W, Shen Y, Wang R, Liu A, Ling H, Li Y, Cao J, Zhang X, Shu J, Zhang L:Cryptosporidium cuniculus and Giardia duodenalis in rabbits: genetic diversity and possible zoonotic transmission. PLoS ONE. 2012, 7: e31262-10.1371/journal.pone.0031262.PubMed CentralView ArticlePubMedGoogle Scholar
- Chalmers RM, Elwin K, Hadfield SJ, Robinson G: Sporadic human cryptosporidiosis caused by Cryptosporidium cuniculus, United Kingdom, 2007-2008. Emerg Infect Dis. 2011, 17: 536-538. 10.3201/eid1703.100410.PubMed CentralView ArticlePubMedGoogle Scholar
- Shi K, Jian F, Lv C, Ning C, Zhang L, Ren X, Dearen TK, Li N, Qi M, Xiao L: Prevalence, genetic characteristics, and zoonotic potential of Cryptosporidium species causing infections in farm rabbits in China. J Clin Microbiol. 2010, 48: 3263-3266. 10.1128/JCM.00819-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu X, Zhou X, Zhong Z, Chen W, Deng J, Niu L, Wang Q: New subtype of Cryptosporidium cuniculus isolated from rabbits by sequencing the gp60 gene. J Parasitol. 2014, 100: 532-536. 10.1645/13-223.1.View ArticlePubMedGoogle Scholar
- Ryan UM, Xiao L, Read C, Zhou L, Lal AA, Pavlasek I: Identification of novel Cryptosporidium genotypes from the Czech Republic. Appl Environ Microbiol. 2003, 69: 4302-4307. 10.1128/AEM.69.7.4302-4307.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Molloy SF, Smith HV, Kirwan P, Nichols RA, Asaolu SO, Connelly L, Holland CV: Identification of a high diversity of Cryptosporidium species genotypes and subtypes in a pediatric population in Nigeria. Am J Trop Med Hyg. 2010, 82: 608-613. 10.4269/ajtmh.2010.09-0624.PubMed CentralView ArticlePubMedGoogle Scholar
- Laboratory-based surveillance for Cryptosporidium in France, 2006-2009. Euro Surveillance. 2010, 15: 19642-Google Scholar
- Kaupke A, Kwit E, Chalmers R, Michalski M, Rzeżutka A: An outbreak of massive mortality among farm rabbits associated with Cryptosporidium infection. Res Vet Sci. 2014, 97: 85-87. 10.1016/j.rvsc.2014.04.016.View ArticlePubMedGoogle Scholar
- Nolan MJ, Jex AR, Haydon SR, Stevens MA, Gasser RB: Molecular detection of Cryptosporidium cuniculus in rabbits in Australia. Infect Genet Evol. 2010, 10: 1179-1187. 10.1016/j.meegid.2010.07.020.View ArticlePubMedGoogle Scholar
- Nolan MJ, Jex AR, Koehler A, Haydon SR, Stevens MA, Gasser RB: Molecular-based investigation of Cryptosporidium and Giardia from animals in water catchments in southeastern Australia. Water Res. 2013, 47: 1726-1740. 10.1016/j.watres.2012.12.027.View ArticlePubMedGoogle Scholar
- Triggs B: Tracks, Scats and Other Traces: a Field Guide to Australian Mammals. 2004, Oxford University Press, South MelbourneGoogle Scholar
- Koehler AV, Whipp M, Hogg G, Haydon SR, Stevens MA, Jex AR, Gasser RB: First genetic analysis of Cryptosporidium from humans from Tasmania, and identification of a new genotype from a traveller to Bali. Electrophoresis. 2014, 35: 2600-2607. 10.1002/elps.201400225.View ArticlePubMedGoogle Scholar
- Bowman DD, Georgi JR: Georgis’ Parasitology for Veterinarians. 2009, St. Louis, Saunders/ElsevierGoogle Scholar
- Cooke BD: Rabbits: manageable environmental pests or participants in new Australian ecosystems?. Wildl Res. 2012, 39: 279-289. 10.1071/WR11166.View ArticleGoogle Scholar
- Yoder JS, Beach MJ:Cryptosporidium surveillance and risk factors in the United States. Exp Parasitol. 2010, 124: 31-39. 10.1016/j.exppara.2009.09.020.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.