Experimental Parasitology 125 (2010) 348–356
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Longitudinal multi-locus molecular characterisation of sporadic Australian human clinical cases of cryptosporidiosis from 2005 to 2008 Josephine Ng a, Brian MacKenzie b, Una Ryan a,* a b
Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia PathWest Laboratory Medicine WA, Locked Bag 2009, Nedlands, WA 6909, Australia
a r t i c l e
i n f o
Article history: Received 9 November 2009 Received in revised form 26 February 2010 Accepted 26 February 2010 Available online 4 March 2010 Keywords: Cryptosporidium Australia Humans Cryptosporidiosis 18S rRNA gp60 Multilocus sequence typing Longitudinal survey
a b s t r a c t Cryptosporidium is a gastrointestinal parasite that is recognised as a significant cause of non-viral diarrhea in both developing and industrialised countries. In the present study, a longitudinal analysis of 248 faecal specimens from Australian humans with gastrointestinal symptoms from 2005 to 2008 was conducted. Sequence analysis of the 18S rRNA gene locus and the 60 kDa glycoprotein (gp60) gene locus revealed that 195 (78.6%) of the cases were due to infection with Cryptosporidium hominis, 49 (19.8%) with Cryptosporidium parvum and four (1.6%) with Cryptosporidium meleagridis. A total of eight gp60 subtype families were identified; five C. hominis subtype families (Ib, Id, Ie, If and Ig), and two C. parvum subtype families (IIa and IId). The Id subtype family was the most common C. hominis subtype family identified in 45.7% of isolates, followed by the Ig subtype family (30.3%) and the Ib subtype family (20%). The most common C. parvum subtype was IIaA18G3R1, identified in 65.3% of isolates. The more rare zoonotic IId A15G1 subtype was identified in one isolate. Statistical analysis showed that the Id subtype was associated with abdominal pain (p < 0.05) and that in sporadic cryptosporidiosis, children aged 5 and below were 1.91 times and 1.88 times more likely to be infected with subtype Id (RR 1.91; 95% CI, 1.7–2.89; p < 0.05) and Ig (RR 1.88; 95% CI, 1.10–3.24; p < 0.05) compared to children aged 5 and above. A subset of isolates were also analysed at the variable CP47 and MSC6-7 gene loci. Findings from this study suggest that anthroponotic transmission of Cryptosporidium plays a major role in the epidemiology of cryptosporidiosis in Western Australian humans. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Cryptosporidiosis is a gastrointestinal disease in humans and animals caused by the protozoan parasite Cryptosporidium. The disease, characterised by self-limiting diarrhoea in immunocompetent individuals, may be chronic and life-threatening to those that are immunocompromised (Hunter and Nichols, 2002; Hunter et al., 2007). Of the 21 valid Cryptosporidium species, Cryptosporidium hominis and Cryptosporidium parvum are responsible for the majority of infections in humans, although Cryptosporidium meleagridis Cryptosporidium felis and Cryptosporidium canis and to a lesser extent, Cryptosporidium muris, Cryptosporidium suis and several genotypes have also been reported in humans (Xiao and Fayer, 2008; Xiao, 2010). Oocysts of most Cryptosporidium spp. are morphologically similar and therefore they can only be distinguished by molecular means, usually at conserved loci such as the 18S rRNA or the actin
* Corresponding author. Address: Division of Health Sciences, School of Veterinary and Biomedical Science, Murdoch University, Murdoch, WA 6150, Australia. Fax: +61 8 9310 414. E-mail address:
[email protected] (U. Ryan). 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.02.017
gene. Several fingerprinting tools have been developed to examine the population structure and transmission dynamics of C. parvum and C. hominis including sequencing of the 60-kDa glycoprotein (gp60) gene, which has shown to be useful in tracking source of infection of C. parvum and C. hominis (Strong et al., 2000; Mallon et al., 2003a; Peng et al., 2003a; Widmer et al., 2004; Xiao and Ryan, 2004; Sulaiman et al., 2005; Alves et al., 2006; Feltus et al., 2006; Gatei et al., 2006; Trotz-Williams et al., 2006; Leoni et al., 2007; Thompson et al., 2007; Ng et al., 2008; O’Brien et al., 2008). Cryptosporidiosis was listed as a nationally notifiable disease in Australia in 2001 (Communicable Disease Surveillance: Highlights for 2nd quarter 2001, 2001) and since then, incidence of the disease in Australia has been increasing steadily with 2009 recording the highest number of cases nationally compared to that of the previous 5 years (National Notifiable Diseases Surveillance System (NNDSS), http://www.health.gov.au). In Western Australia (WA), 625 cases of cryptosporidiosis were notified in 2007, which was almost three times that of notification rates for the previous 4 years. Molecular studies of Cryptosporidium species infecting humans in WA, New South Wales (NSW), Victoria (VIC) and South Australia (SA) have identified three Cryptosporidium species; C. hominis, C. parvum and C. meleagridis with C. hominis being the most
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frequently identified species of the three (Robertson et al., 2002; Chalmers et al., 2005; Jex et al., 2007, 2008; Ng et al., 2008; O’Brien et al., 2008; Alagappan et al., 2009; Waldron et al., 2009a,b). The published studies to date however were conducted on limited numbers of sporadic cases of cryptosporidiosis and no longitudinal molecular studies of cryptosporidiosis have been conducted in Australia. The aim of the present study therefore, was to examine samples from sporadic cases of cryptosporidiosis in WA humans over a 3-year period (May 2005–March 2008) to identify the species and subtypes of Cryptosporidium causing sporadic human cryptosporidiosis to better understand the transmission dynamics and distribution of the parasite in WA.
The confidence of groupings was assessed by bootstrapping, using 1000 replicates.
2. Materials and methods
3.1. Cryptosporidium species
2.1. Specimens and DNA extraction
From the 254 samples received between May 2005 and March 2008, 248 samples were amplified at the 18S rRNA locus. Analysis identified 195/248 (78.6%) as C. hominis, 49/248 (19.8%) as C. parvum and 4/248 (1.6%) as C. meleagridis (Table 1). Of the 77 positive specimens received in 2005/2006, 74 were amplified at the 18S rRNA gene locus. C. hominis was identified in 62.2% of the isolates, C. parvum in 33.8% and C. meleagridis in 4.1% as (Table 1). Infection with C. hominis was the highest amongst patients aged 0–5 years in 87.2% of cases, compared to infection C. parvum and C. meleagridis, in 10.3% and 2.6% of cases, respectively (Table 2). Cases of Cryptosporidium were more prevalent in regional areas of Western Australia at 81.2% compared to urban areas at 18.8%, of which C. hominis was identified in 90.9% of these cases (Table 3). In 2006/2007, a total of 107/109 isolates received were amplified at the 18S rRNA gene locus, with 90.7% of the isolates identified as C. hominis, 8.4% as C. parvum and 0.9% as C. meleagridis (Table 1). C. hominis was most prevalent across all age groups, of which 60.2% of cases were from the age group 0–5, 21.2% from 16 to 39 years and 6.8% and 3.9%, respectively, for 6–15 and >40 years. The single C. meleagridis case identified, was from the 16–39 age group (Table 2). There were a higher number of Cryptosporidium cases from regional areas of Western Australia (80.6%), compared to urban areas (19.4%), with C. hominis infection identified in 85% of urban and 94% of regional cases, respectively (Table 3). From the 68 isolates received in 2007/2008, 67 isolates successfully amplified at the 18S rRNA gene locus, of which C. hominis was identified in 77.6% of the samples and C. parvum in 22.4% of the samples. No C. meleagridis were identified from specimens received in this year. Most cases (60.7%) were from patients aged 5 and below and of these, 94.1% were C. hominis. The majority of specimens received were from regional areas, with C. hominis being more prevalent than C. parvum in regional areas (90% of cases) (Table 3).
A total of 254 microscopy positive human faecal specimens from sporadic cases of cryptosporidiosis were collected from various pathology centres in WA from May 2005 to March 2008. All samples were stored at 4 °C prior to molecular analysis. Patient epidemiological information for most of the specimens were collected (age, n = 234; location, n = 230; symptoms or clinical signs, n = 209; collection date, n = 234). Total DNA was extracted using a QIAmp DNA Stool Kit (Qiagen, Germany). 2.2. PCR amplification and DNA sequencing Initial genotyping of the samples were carried out by PCR-RFLP of an 830 bp fragment of the Cryptosporidium 18S gene locus as described by Xiao et al. (2001), using restriction analysis of the PCR product by the VspI (Promega, USA) to discriminate between C. hominis and C. parvum. For samples that failed to amplify or produced ambiguous banding patterns, a two-step nested PCR and sequencing of a 540 bp product the 18S gene locus was carried out (Ryan et al., 2003). Cryptosporidium hominis and C. parvum positive samples were sub-typed at the gp60 gene locus using a two-step nested PCR that amplifies a 830 bp fragment (Strong et al., 2000; Sulaiman et al., 2005). Secondary PCR products were purified and sequenced as described by Ng et al. (2006). Multilocus sequence typing was carried out on a smaller number (n = 13) of C. hominis and C. parvum positive clinical human isolates at the MSC6-7 gene locus (serine repeat antigen) and the CP47 gene locus (47-kDa protein) and subtypes were assigned according to Gatei et al. (2007). Briefly, at the MSC6-7 gene locus, subtypes were assigned based on the minisatellite TGATGATGAT(G)GAACC(T) in the repeat region and single nucleotide polymorphisms (SNPs) in the non-repeat region. For the CP47 gene locus, C. hominis was assigned as type I and classified based on the number of TAA repeats, (coded as A) and TGA/TAG repeats, (coded as G), with the following digit showing the number of trinucleotide repeats (Gatei et al., 2007). Isolates were selected for analysis at the MSC6-7 and CP47 gene loci based on their gp60 subtype family; Ib (1), Id (3), Ie (2), Ig (3) and four IIa subtypes, A17G2R1 (1), A18G3R1 (2) and A19G4R1 (1). Amplification of both gene loci was carried out as described in Gatei et al. (2006). Nucleotide sequences were analyzed using ChromasPro version 2.3 (http://www.technelysium.com.au) and aligned using ClustalW (http://clustalw.genome.jp). Distance estimation was conducted using TREECON (Van de Peer and De Wachter, 1994), based on evolutionary distances calculated with the Kimura-2 model and grouped using neighbour joining. Parsimony analysis was conducted using MEGA version 3.1 (MEGA3.1: Molecular Evolutionary Genetics Analysis software, Arizona State University, Tempe, AZ).
2.3. Statistical analysis Statistical analysis was performed using SPSS 17.0 (Statistical Package for the Social Sciences) for Macintosh OS X (SPSS Inc. Chicago, USA) to determine if there was any association between subtypes and within subtype families versus age, gender and clinical symptoms. 3. Results
3.2. Gp60 sub-genotyping of C. hominis and C. parvum A total of 243 isolates, previously typed at the 18S locus, were successfully sub-typed at the gp60 locus. Representative sequences from each subtype were deposited in GenBank under the Accession Nos. GU933438–GU933458. The four C. meleagridis and one C. hominis isolates failed to amplify at the gp60 gene locus. A total of five C. hominis subtype families, Ib (39/243), Id (88/243), Ie (4/243), If (3/243) and Ig (60/243), and two C. parvum subtype families, IIa (48/243) and IId (1/243) were identified. Within the subtype families, two Id subtypes (IdA15G1 and IdA16), two Ig subtypes (IgA17 and IgA19) and five different IIa subtypes (IIaA17G2R1, IIaA18G3R1, IIaA19G4R1, IIaA20G3R1 and IIaA21G3R1) were identified (Table 4 and Fig. 1a). The most common C. hominis subtype identified was the IdA15G1 subtype in 45.1% of the C. hominis
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Table 1 Cryptosporidium species identified per yearly range from May 2005 to March 2008. Yearly range May 2005–March 2006
April 2006–March 2007
April 2007–March 2008
C. hominis C. parvum C. meleagridis
46 25 3
97 9 1
52 15 0
Total
74
107
67
Table 4 Cryptosporidium hominis and C. parvum GP60 subtypes identified in Western Australian humans from 2005 to 2008. Subtype family C. hominis Ib Id Ie If Ig
Table 2 Distribution of Cryptosporidium species in Western Australian humans by age. Age range (years)
%
Species C. hominis
C. parvum
C. meleagridis
54.9 18.3 14.1 12.7
34 6 3 1 44
4 6 6 5 21
1 0 1 1 3
2006/2007 (n = 103 ) 0.05).
4. Discussion Results revealed that anthroponotic transmission of Cryptosporidium is common amongst WA humans, similar to reports from previous studies where the prevalence of C. hominis in Cryptosporidium positive samples from WA was 83% and 82% (Morgan et al., 1998; O’Brien et al., 2008). Findings from the present study also showed that C. hominis was most prevalent followed by C. parvum and C. meleagridis, corresponding to reports from studies conducted in SA, VIC and NSW which have also reported C. hominis as the most common species in sporadic cryptosporidiosis cases (Chalmers et al., 2005; Jex et al., 2007, 2008; Pangasa et al., 2009; Waldron et al., 2009b). Previous studies have reported that C. hominis was more prevalent in urban areas and C. parvum was more prevalent in regional areas (McLauchlin et al., 2000; Learmonth et al., 2004; Feltus et al., 2006; Zintl et al., 2009). Recent studies in NSW, reported that C. parvum was detected in higher
proportions in sporadic cryptosporidiosis cases in regional NSW and in urban areas in and around Sydney, with similar prevalence of 57% (Ng et al., 2008; Alagappan et al., 2009), whereas, in another study on sporadic cases in NSW, equal distributions of C. hominis and C. parvum among urban and regional areas was reported (Waldron et al., 2009a). In the present study however, C. hominis was found to be much more prevalent than C. parvum in both urban and regional WA, suggesting that there may be distinct geographical differences in the prevalence and transmission of C. hominis and C. parvum within Australia. The zoonotic C. meleagridis is recognised as the third most common Cryptosporidium species found in humans, and in some places, is found to be as prevalent as C. parvum (Xiao and Feng, 2008). C. meleagridis is usually identified in children and immunocompromised adults from developing and industrialised countries (Morgan et al., 2000; Guyot et al., 2001; Gatei et al., 2002; Alves et al., 2006). In the present study, C. meleagridis was identified in 4/248 of the WA clinical isolates. Of these, one was from a 3-year old child and three were from the 18–40 years age range, one of which was from a patient with chronic myeloid leukaemia. Epidemiological data showed reports of diarrhoea in all four cases, with one adult experiencing diarrhoea and fever. C. meleagridis has been reported in a wide range of avian hosts (Ryan, 2010), but was not identified in a study of Cryptosporidium species in birds in WA (Ng et al., 2006). Hence, although birds may play a role in the transmission of C. meleagridis, human to human transmission appears to be the more likely transmission mode. C. meleagridis has been identified in two separate studies of sporadic cases of cryptosporidiosis in VIC (Jex et al., 2007; Pangasa et al., 2009).
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Seasonal trends in the distribution of C. hominis and C. parvum cases have been reported in the United Kingdom and New Zealand, with cases of cryptosporidiosis reported in spring mostly due to C. parvum and cases reported in autumn mainly due to C. hominis infection (McLauchlin et al., 2000; Goh et al., 2004). In the present study, no definite trends were observed, with the number of cases due to C. hominis more prevalent than C. parvum across all seasons from 2005 to 2008 (Fig. 2). The number of cases appeared to increase from spring and peaked in autumn with a pronounced peak observed in the autumn of 2007 following an outbreak (Ng et al., 2010). An equal proportion of IbA10G2 and IdA15G1 gp60 subtypes were identified from cases in autumn 2007 (Table 5). However, in the present study, the majority of isolates typed were from rural locations whereas genotyping of the urban-derived Cryptosporidium isolates attributed to the 2007 outbreak were mostly identified as the IbA10G2 subtype (Ng et al., 2010). Cryptosporidium hominis and C. parvum heterogeneity was observed at the gp60 gene locus with 14 subtypes from 7 subtype families identified among positive WA human isolates (Table 4). The Ib subtype family has been reported as the most common C. hominis subtype identified in Australia (Jex et al., 2007, 2008; Waldron et al., 2009a,b). However, in the present study, the Id subtype family was the most dominant subtype identified. The Id subtype family has been reported in developing countries such as South Africa, China, India and Malawi (Leav et al., 2002; Peng et al., 2001, 2003b; Gatei et al., 2007). There appeared to be an association between the two most common subtypes identified in the present study; IdA15G1 and IgA17, in individuals aged 5 years and below Previous studies have shown that infection with the Id subtype family was associated with general and chronic diarrhoea and was the most virulent subtype in HIV-infected patients (Cama et al., 2007, 2008). In the present study, among those infected with the Id subtype family, 84.4% (54/64) reported diarrhoea with no indication of the length or severity and 7.8% (5/64) of cases reported diarrhoea and vomiting. In general, diarrhoea was the most common clinical symptom reported, in all subtype families identified, except for the Ie and IId subtype families where no data for clinical symptoms were available. Data for clinical symptoms in the present study was however inadequate to generate statistically significant associations within subtype families and clinical symptoms. Future studies should in-
clude consistent description of symptoms or case definitions, which will allow for better categorisation of data. For those infected with IgA17, 87% (40/46) of cases reported diarrhoea with 8.7% (4/46) of cases reported diarrhoea and vomiting. The severity and length of these symptoms however, were not available. The prevalence of Ig was consistent in 2005/06 and 2006/ 07 at 31% and 30.5%, respectively, but for 2007/08, the prevalence of Ig was only 9.0%. The numbers of the Ig subtype family identified in samples received were fairly consistent across the seasons, peaking in autumn 2006. However, from summer 2007, there was an increase in the number of Ib and Id isolates identified, which peaked in equal numbers in autumn 2007, following the WA outbreak (Ng et al., 2010). The increase in the presence of the more virulent subtype families, such as the Ib subtype family, has been associated with a number of waterborne outbreaks worldwide (Zhou et al., 2003; Cohen et al., 2006; Leoni et al., 2007). The Ig subtype family has previously been reported in sporadic cases of cryptosporidiosis during a drinking-water associated outbreak in Northern Ireland (Glaberman et al., 2002) but was not identified in a more recent study on the prevalence of Cryptosporidium in humans in Ireland (Zintl et al., 2009). Subtyping studies have identified some C. parvum subtypes that have only been found in humans (Sulaiman et al., 2005; Alves et al., 2006; Trotz-Williams et al., 2006), refuting previous assumptions that all C. parvum infections in humans were due to zoonotic transmission. The two most common C. parvum subtypes families identified are the IIa subtype family, which have been identified in humans and ruminants and are responsible for the majority of zoonotic transmission in sporadic cases of cryptosporidiosis in humans, and the IIc subtype family, which has so far only been identified in humans (Xiao, 2010). In the present study, all cases with C. parvum were attributed to infection with the IIa subtype family, except for one case where the IId subtype was identified (Table 4). Within the IIa subtype family, five subtypes were identified, of which the IIaA18G3R1 subtype was the most common subtype identified, in 66.7% of cases with IIa infection (Table 4). The IIaA18G3R1 subtype is one of the most commonly reported C. parvum subtype in humans and cattle in Australia and Ireland (Thompson et al., 2007; O’Brien et al., 2008; Ng et al., 2008; Jex et al., 2008; Zintl et al., 2009; Waldron et al., 2009a,b). The other subtypes identified in the present study;
C. hominis
No. of C. hominis / C. parvum identified
60
250
C. parvum Total no. of cases
50
200 40 150 30 100 20 50
10
0
0 Autumn, May 2005
Winter 2005
Spring 2005
Summer 2005/06
Autumn 2006
Winter 2006
Spring 2006 Seasons
Summer 2006/07
Autumn 2007
Winter 2007
Spring 2007
Summer 2007/08
Autumn, March 2008
Fig. 2. Seasonal distribution of C. hominis and C. parvum and the total number of cases reported in Western Australia in 2005–2008.
Total no. of reported cases in Western Australia
300
70
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Table 5 Seasonal distribution of Cryptosporidium GP60 subtype families and subtypes in Western Australia humans. Months of the season in Australia – Summer, December–February, Autumn, March–May, Winter, June–August, Spring, September–November. Subtype family
Subtype
Autumn, May 2005
Winter 2005
Spring 2005
Summer 2005/06
Autumn 2006
Winter 2006
Spring 2006
Summer 2006/07
Autumn 2007
Winter 2007
Spring 2007
Summer 2007/08
Autumn, March 2008
C. hominis (n = 195)a Ib A10G2 Id A15G1 A16 Ie A11G3T3 A12G3T3 If A12G1 Ig A17 A19
– 5 – – – – 1 –
– 3 – – – – 7 –
– 2 – – – – 6 –
1 5 – 1 – 2 7 –
3 7 – 2 – – 16 –
1 3 – – – – 2 –
1 9 – – – – 4 –
3 15 2 – – 1 6 –
28 28 – – – – 7 –
1 1 – – – – 1 –
1 3 – – – – 1 –
– 5 – – – – 1 –
– – – – 1 – – 1
C. parvum (n = 49) IIa A17G2R1 A18G3R1 A19G4R1 A20G3R1 A21G2R1 IId A15G1
– 1 – – 1 –
4 6 – – – –
1 1 – – – –
3 6 1 – – –
– 2 2 – – –
1 4 – – – –
– 1 – – – –
– – – – – –
– 2 – – – –
– – – – – –
– 4 – 1 – –
2 4 – – – 1
– 1 – – – –
–
1
1
1
–
1
–
–
–
–
–
–
–
C. meleagridis (n = 4) a
One isolate, positive at the 18S rRNA gene locus did not amplify at the GP60 gene locus.
IIaA17G2R1, IIaA20G3R1 and IIaA21G2R1, were also detected in NSW cattle and cattle in Ireland (Thompson et al., 2007; Ng et al., 2008), implicating the importance of the role of ruminants in the zoonotic transmission of the C. parvum IIa subtype family. The IId subtype family is relatively rare but has been reported in humans and cattle in Portugal (Alves et al., 2003, 2006), cattle in Hungary (Plutzer and Karanis, 2007), lambs and goats in Spain (Quilez et al., 2008) and humans in Ireland, Kuwait, the Netherlands and Australia (Sulaiman et al., 2005; Wielinga et al., 2008; Zintl et al., 2009; Waldron et al., 2009b). IId subtypes of C. parvum have never been found in humans or calves in the United States and Canada (Xiao, 2010). In the present study, the one IId subtype identified, IIdA15G1, was from a 3-year old from an urban area in WA. To date, no subtypes from the IId family have been identified in any animals in Australia. In the Netherlands, the IId subtype family, including the IIdA15G1 subtype, was only identified in humans and not in farm animals (Wielinga et al., 2008). More genotyping studies need to be carried out in order to understand and assess the transmission route for this subtype. Across Australia, subtyping of the gp60 gene has been used to study cases of sporadic human and cattle cryptosporidiosis in four different states (WA, SA, VIC and NSW). These studies have shown that Australia harbours a total of six different C. hominis gp60 subtype families and only three different C. parvum gp60 subtype families with WA and NSW showing higher subtype heterogeneity than VIC and SA (Table 6). Up to 15 subtypes from six different gp60 subtype families have been identified in WA whilst NSW have reported 12 subtypes from five different subtype families. Within Australia, large subtype heterogeneity has been observed in C. hominis gp60 Ib and Id subtype families, with eight and six subtypes in each respective family. For the Ib subtype family however, only the IbA10G2 subtype was identified across the four different states with a prevalence ranging from 16% (in WA) to 97.3% (in VIC) and has been implicated as the cause of a recent outbreak in NSW (Waldron and Power, 2009). Three different C. parvum subtype families have been identified in Australia; IIa, IIc and IId. Both the IIa subtype family (which was the most common subtype identified across all states) and the anthroponotic IIc subtype family have been reported in all four states, whereas the IId subtype family has only been identified in WA (1 IIdA15G1 subtype) and NSW (1 IIdA24G1 subtype) (Table 6). The IIa subtype family exhibited the highest level of genetic heterogeneity with a total of 16 differ-
ent subtypes identified across Australia. Large heterogeneity amongst IIa subtypes however, is not uncommon and has been observed in studies of C. parvum infected humans in Ireland and cattle in the Netherlands (Wielinga et al., 2008; Zintl et al., 2009). IIaA18G3R1 is the most common IIa subtype and has been identified in cases from humans in WA, SA, VIC and NSW and cases from cattle in WA and NSW. Other IIa subtypes such as IIaA15G2R1, IIaA16G3R1, IIaA17G2R1, IIaA19G4R1 and IIaA20G3R1 have been identified in both human and cattle cases in Australia, implicating the potential role of cattle in the zoonotic transmission of C. parvum. Multilocus sequence typing (MLST) and multilocus typing (MLT) studies have identified distinct C. hominis subtypes from different geographical locations and the type of Cryptosporidium populations within countries (Mallon et al., 2003a,b; Ngouanesavanh et al., 2006; Gatei et al., 2006, 2007, 2008). In the present study, two additional gene loci were analysed; CP47 and MSC6-7. The CP47 locus is a 47 kDa protein gene and subtypes are defined based on differences within its microsatellite region. The MSC6-7 locus is a serine repeat antigen gene and typing is based on SNP differences in minisatellite repeats and in the non-minisatellite region. Both these genes have previously been shown to exhibit high DNA sequence diversity and have been used to identify genetic differences between C. hominis isolates from different countries (Gatei et al., 2006, 2007, 2008). In the present study, sequence typing and phylogenetic analysis showed that all gp60 subtypes analysed could be differentiated into corresponding CP47 subtypes, with the exception of two gp60 IeA11G3T3 subtype isolates (H232 and H260), which formed two different CP47 subtypes (Fig. 1b). At the MSC6-7 gene locus however, phylogenetic analysis showed that all three IgA17 subtype isolates (H172, H196, H226) formed a single group, although based on the MSC6-7 sequence length polymorphism, a 15 bp length variation was noted for one of the three IgA17 subtype isolate (H172). Interestingly, two very different gp60 subtype families; isolates H209 (gp60 subtype IbA10G2) and H250 (gp60 subtype IdA15G1) grouped together at the MSC6-7 and were separate from all other C. hominis isolates (Fig. 1c). Application of the MLST tool may be useful in elucidating and comparing the population structures and transmission of Cryptosporidium from different geographical locations. There is however, a paucity of sequences for the CP47 and MSC6-7 loci in GenBank, which makes compari-
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Table 6 Summary of GP60 subtypes identified in humans and cattle in Australia and the number of isolates identified per species in each study. Host Human
Species C. hominis
Sub-subtypes
Western Australia
New South Wales 6
Victoria
South Australia
IaA10R4 IaA17R1 IaA17R3 IaA23 IbA5G2T3 IbA6G3 IbA9G2 IbA9G2T1 IbA9G2 IbA9G3 IbA10G2 IbA18G1 IdA15 IdA15G1 IdA16 IdA24T1 IdA25 IdA26 IeA11G3T3 IeA12G3T3 IfA11G1T1 IfA12G1 IgA17 IgA19
– – 1/415 1/415 – 1/415 1/415 1/415 – 21/415; 40/1977 – – 9/415; 88/1977 1/415; 2/1977 – 3/415 – 3/1977 1/1977 1/415 2/415; 4/1977 58/1977 1/1977
1/37 – 1/376 – – 1/376 2/376 – 2/376 – 2/234; 25/376 – 1/376 1/374 – 1/376 – 1/376 1/376 – – 1/376 – –
– – – – – – – – – – 72/742 1/742 – 1/742 – – – – – – – – – –
– 3/383 – – – – – – – 7/221; 14/383 20/383 – – – – – – – – – – 1/383 – –
Human
C. parvum
IIaA15G2R1 IIaA16G3R1 IIaA16G4R1 IIaA17G2R1 IIaA17G3R1 IIaA17G4R1 IIaA18G3R1 IIaA19G3R1 IIaA19G4R1 IIaA20G2R1 IIaA20G3R1 IIaA20G5R1 IIaA21G2R1 IIaA22G3R1 IIaA22G4R1 IIaA23G3R1 IIcA5G3a IIdA15G1 IIdA24G1
2/75 – – 1/75; 11/497 – – 3/75; 32/497 1/75 1/75; 3/497 – 1/497 – 1/497 – – – 1/75 1/497 –
1/326 2/326 1/326 1/44; 1/326 1/326 1/326 1/44; 10/326 1/44; 1/326 – 1/326 1/44; 1/326 3/326 – 1/326 – – 1/76 – 1/326
– – – – – – 9/232 – – 7/232 – – – 1/232 – 1/232 5/232 – –
– – – – – – 15/243 – – 6/243 – – – – 2/243 – 1/243 – –
Cattle
C. parvum
IIaA15G2R1 IIaA16G3R1 IIaA17G2R1 IIaA18G3R1 IIaA19G4R1 IIaA20G3R1 IIaA21G3R1
2/75 – – 5/75 – – –
– 3/134 1/134 5/134 2/134 1/134 1/134
– – – – – – –
– – – – – – –
Superscript denotes the reference study where the subtypes were identified: 1Chalmers et al. (2005); 2Jex et al. (2007); 3Jex et al. (2008); 4Ng et al. (2008); 5O’Brien et al. (2008); 6Waldron et al. (2009b); 7The present study. NB: The gp60 subtype nomenclature used by Jex et al. (2007, 2008) includes some non-serine-coding sequences in the microsatellite repeat number counting, which has caused confusion and is not generally accepted by other researchers. Therefore, the data from SA and Victoria in this table are based on the accepted subtype nomenclature.
son between gp60 subtypes and CP47 and MSC6-7 subtypes difficult. Increased application of these MLST tools will lead to more sequences being deposited in GenBank, which will be useful for future transmission and population studies of this ubiquitous parasite. Anthroponotic transmission of C. hominis among humans is the main mode of transmission in WA as it was the dominant species identified in isolates from both urban and rural areas of WA. Regular surveillance of cryptosporidiosis in rural regions may assist in understanding the transmission dynamics of C. hominis in these areas and the implementation of better control measures. Zoonotic transmission of Cryptosporidium, however, should not be underestimated. Cattle may play an important role in the transmission of C. parvum as C. parvum gp60 subtypes, which have been previously reported in cattle, were identified in humans in the present study.
Larger studies of Australian cattle and sheep populations are required to determine the extent of their role in zoonotic transmission of Cryptosporidium. Acknowledgments This study was funded by an ARC-Linkage Grant (LP0561862) and the support of the Sydney Catchment Authority (SCA). The authors gratefully acknowledge the participation of PathWest Laboratory Medicine WA. References Alagappan, A., Tujula, N.A., Power, M., Ferguson, C.M., Bergquist, P.L., Ferrari, B.C., 2009. Development of fluorescent in situ hybridization for Cryptosporidium
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