Wildlife-associated Cryptosporidium fayeri in Human, Australia

To the Editor: Molecular tools are essential for Cryptosporidium spp. identification, taxonomy, and epidemiology because of morphologic similarities between species within this genus. Molecular analyses have now identified 22 Cryptosporidium spp. and >40 cryptic species (i.e., genotypes) across all vertebrate classes (1). The myriad of potential Cryptosporidium spp. hosts, in conjunction with the robustness of the infectious stage (oocyst), means diverse Cryptosporidium spp. constantly circulate through the environment. This circulation increases the potential for disease from a diversity of contamination sources. 
 
Human cryptosporidiosis is a global problem causing illness in young, elderly, immunocompromised, and immunocompetent persons in both industrialized and developing nations. The 2 most common etiologic agents, responsible for 90% of reported human infections, are C. hominis and C. parvum (2,3). Additional species identified as human pathogens are C. meleagridis, C. canis, C. felis, and the Cryptosporidium rabbit genotype (4). Each of these species was once thought to be specific for turkeys, dogs, cats, and rabbits, respectively. Incidental findings of C. muris, C. andersoni, C. suis, C. hominis monkey genotype, C. parvum mouse genotype, and Cryptosporidium cervine (W4), chipmunk I (W17), skunk, and horse genotypes have also been reported in humans (4). The pathogenicity of these zoonotic species and genotypes to humans remains unclear. 
 
In July 2009, a 29-year-old woman who sought care because of prolonged gastrointestinal illness had a fecal test positive for Cryptosporidium spp. by the Remel ProSpecT Giardia/Cryptosporidium microplate assay (Thermo Fisher Scientific, Lenexa, KS, USA). Oocysts were purified from the specimen (5) and stained with the Cryptosporidium spp.–specific antibody CRY104 labeled with fluorescein isothiocyanate (Biotech Frontiers, North Ryde, Australia) for enumeration. A parasite load of 1.34 × 106 oocysts/g feces was determined by using epifluorescence microscopy at 400× magnification. 
 
To identify Cryptosporidium spp., DNA was extracted (5), and a diagnostic fragment of the small subunit (SSU) rRNA) was amplified (6). Clones were screened to identify species and determine the possibility of mixed infection. Plasmids from 50 clones were recovered and digested with the enzyme SspI (New England Biolabs, Beverly, MA, USA) (6). Two different restriction profiles were visualized. The sequence from each of the restriction types was determined; profile 1 contained SspI fragment sizes of 33, 109, 247, and 441 bp; profile 2 had fragments of 33, 254, and 540bp. A BLAST search (www.ncbi.nlm.nih.gov/blast) confirmed the sequences as C. fayeri type 1 and type 2. These 2 sequences correspond to known heterogeneity within the SSU rRNA of C. fayeri (7). 
 
The identification of C. fayeri by SSU rRNA was confirmed by the sequence of the actin gene (8), showing 99.8% similarity to C. fayeri (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF112570","term_id":"5714417","term_text":"AF112570"}}AF112570). Further analysis at the 60-kDa glycoprotein (gp60) locus was used to determine the Cryptosporidium subtype family (5). The MQ1022 gp60 sequence was 98% similar to C. fayeri subtype family IVa (9). Analysis of the microsatellite region further characterized isolate MQ1022 to C. fayeri subtype IVaA9G4T1R1. The nucleotide sequences generated in this study were submitted to GenBank under accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"HQ008932-HQ008934","start_term":"HQ008932","end_term":"HQ008934","start_term_id":"313506256","end_term_id":"313506259"}}HQ008932-HQ008934. 
 
Because the patient was imunocompetent, the disease was believed to be self-limiting, and she was lost to follow-up. The patient resided in a national forest on the east coast of New South Wales, Australia, an area where marsupials are abundant. She had frequent contact with partially domesticated marsupials. Notably, C. fayeri has been identified in 6 Australian marsupial species. Identification of C. fayeri in a human patient is a concern for water catchment authorities in the Sydney region. The main water supply for Sydney, Warragamba Dam, covers 9,050 km2 and is surrounded by national forest inhabited by diverse and abundant marsupials. A previous study that investigated Cryptosporidium spp. in a wild eastern gray kangaroo (Macropus giganteus) population reported a prevalence of 6.7% (10). Oocyst shedding ranged from 20/g feces to 2.0 × 106/g feces (10). Subtype IVaA9G4T1R1 identified from the patient in this study has been characterized from eastern gray kangaroos in Warragamba Dam (9). Throughout the year, large groups of eastern gray kangaroos graze within riparian zones in the catchment. Such close proximity to the water presents a high possibility that the dam’s water is contaminated with oocysts from these animals. 
 
The Cryptosporidium genus is diverse, both in species and suitable hosts. The mechanisms of host specificity remain unknown, but the frequency of Cryptosporidium spp. crossing the host barrier and becoming zoonoses is increasing. This increase indicates that Cryptosporidium spp. host specificity is not as stringent as previously thought.

In July 2009, a 29-year-old woman who sought care because of prolonged gastrointestinal illness had a fecal test positive for Cryptosporidium spp. by the Remel ProSpecT Giardia/Cryptosporidium microplate assay (Thermo Fisher Scientifi c, Lenexa, KS, USA). Oocysts were purifi ed from the specimen (5) and stained with the Cryptosporidium spp.-specifi c antibody CRY104 labeled with fl uorescein isothiocyanate (Biotech Frontiers, North Ryde, Australia) for enumeration. A parasite load of 1.34 × 10 6 oocysts/g feces was determined by using epifl uorescence microscopy at 400× magnifi cation.
To identify Cryptosporidium spp., DNA was extracted (5), and a diagnostic fragment of the small subunit (SSU) rRNA) was amplifi ed (6). Clones were screened to identify species and determine the possibility of mixed infection. Plasmids from 50 clones were recovered and digested with the enzyme SspI (New England Biolabs, Beverly, MA, USA) (6). Two different restriction profi les were visualized. The sequence from each of the restriction types was determined; profi le 1 contained SspI fragment sizes of 33, 109, 247, and 441 bp; profi le 2 had fragments of 33, 254, and 540bp. A BLAST search (www.ncbi.nlm.nih. gov/blast) confi rmed the sequences as C. fayeri type 1 and type 2. These 2 sequences correspond to known heterogeneity within the SSU rRNA of C. fayeri (7).
The identifi cation of C. fayeri by SSU rRNA was confi rmed by the sequence of the actin gene (8), showing 99.8% similarity to C. fayeri (GenBank accession no. AF112570). Further analysis at the 60-kDa glycoprotein (gp60) locus was used to determine the Cryptosporidium subtype family (5). The MQ1022 gp60 sequence was 98% similar to C. fayeri subtype family IVa (9). Analysis of the microsatellite region further characterized isolate MQ1022 to C. fayeri subtype IVaA9G4T1R1. The nucleotide sequences generated in this study were submitted to GenBank under accession nos. HQ008932-HQ008934.
Because the patient was imunocompetent, the disease was believed to be self-limiting, and she was lost to follow-up. The patient resided in a national forest on the east coast of New South Wales, Australia, an area where marsupials are abundant. She had frequent contact with partially domesticated marsupials. Notably, C. fayeri has been identifi ed in 6 Australian marsupial species. Identifi cation of C. fayeri in a human patient is a concern for water catchment authorities in the Sydney region. The main water supply for Sydney, Warragamba Dam, covers 9,050 km 2 and is surrounded by national forest inhabited by diverse and abundant marsupials. A previous study that investigated Cryptosporidium spp. in a wild eastern gray kangaroo (Macropus giganteus) population reported a prevalence of 6.7% (10). Oocyst shedding ranged from 20/g feces to 2.0 × 10 6 /g feces (10). Subtype IVaA9G4T1R1 identifi ed from the patient in this study has been characterized from eastern gray kangaroos in Warragamba Dam (9). Throughout the year, large groups of eastern gray kangaroos graze within riparian zones in the catchment. Such close proximity to the water presents a high possibility that the dam's water is contaminated with oocysts from these animals.
The Cryptosporidium genus is diverse, both in species and suitable hosts. The mechanisms of host specifi city remain unknown, but the frequency of Cryptosporidium spp. crossing the host barrier and becoming zoonoses is increasing. This increase indicates that Cryptosporidium spp. host specifi city is not as stringent as previously thought.  (1). The host range for CDV is broad, and infection has been found in several mammalian species of the families Canidae, Mustelidae, Procyonidae, Ursidae, and Viverridae (2).
Stelvio National Park (SNP) encompasses 1,333 km 2 of protected land in Italy and covers 2 regions (Lombardia and Trentino Alto Adige); the Lombardia section of the park covers the northern part of Sondrio Province (Valtellina). SNP is surrounded by other parks (Schweitzer National Park, Adamello, and Adamello-Brenta) to form a large protected area (2,500 km 2 ) in the heart of the Alps mountains. Within SNP, the terrestrial mammals that are susceptible to CDV include red foxes (Vulpes vulpes), stoats (Mustela erminea), weasels (Mustela nivalis), pine martens (Martes martes), beech martens (Martes foina), badgers (Meles meles), and bears (Ursus arctos).
In August 2009, three young red foxes were captured in Valtellina (Sondrio), Lombardia, Italy, within the southwestern borders of SNP. The animals showed canine distemper-like signs (e.g., prostration, altered behavior, and conjunctivitis), and CDV infection was confi rmed by quantitative reverse transcription-PCR of pooled organs (3). In September and October 2009, another 2 young foxes were captured and found to be positive for CDV. From September on, at least 30 foxes with altered behavior were seen near human habitations and facilities in SNP; 10 were captured. In the same period, infected foxes were also reported from Engadina, Switzerland, at the northern and western borders of SNP. In February 2010, two symptomatic foxes were euthanized in Grosotto, 50 km south of where the initial cases were identifi ed. The epizootic appeared to have originated from the eastern regions of Italy (Trentino Alto Adige, and Veneto), where CDV infection had been reported in red foxes and badgers since August 2006 (4) (Figure). A large CDV epidemic in foxes in southern Bavaria in 2008 has also been described, thus suggesting spread of the virus throughout the Alps area (5).
Reverse transcription-PCR genotyping of the hemagglutinin (H) gene (6) identifi ed 15 CDV strains, which were analyzed and characterized as European genotypes. The full-length H gene of the CDV strains was determined (GenBank accession no. HM120874). Sequence analysis of the H gene indicated that the fox CDV