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Applied and Environmental Microbiology, December 2006, p. 7548-7553, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01352-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of Novel Cryptosporidium Genotypes from Avian Hosts

Josephine Ng,^1* Ivan Pavlasek,² and Una Ryan¹

Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia,¹ State Veterinary Institute Prague, Pathology and Parasitology Department, 165 03 Prague 6, Czech Republic²

Received 13 June 2006/ Accepted 25 September 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
A total of 430 avian-derived fecal specimens were randomly collected from selected Western Australian commercial aviaries, poultry farms, hatcheries, wildlife parks, and the Perth Zoo and screened for the presence of Cryptosporidium by PCR. Of these, 27 Cryptosporidium-positive isolates were detected, characterized, and compared with 11 avian-derived isolates from the Czech Republic at the 18S rRNA and actin gene loci. Sequence and phylogenetic analysis identified four genetically distinct genotypes, avian genotypes I to IV, from various avian hosts. In addition, the host range for Cryptosporidium galli was extended. Cryptosporidium muris and Cryptosporidium andersoni were also identified in a tawny frogmouth and a quail-crested wood partridge, respectively.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cryptosporidiosis is one of the most prevalent parasitic infections in domesticated, caged, and wild birds (33), and the parasite has been reported in more than 30 avian species worldwide, belonging to orders Anseriformes, Charadriiformes, Columbiformes, Galliformes, Passeriformes, Psittaciformes, and Struthiniformes (5, 6, 9, 10, 13, 14, 15, 16, 17, 20, 22, 23, 28, 31, 32, 35, 38, 39). However, few studies have examined the genetic diversity of Cryptosporidium sp. among avian hosts.

There are currently three accepted avian species of Cryptosporidium, C. baileyi, C. meleagridis, and C. galli, based on biological and genetic differences (3, 24, 25, 26, 29). Of these, only C. meleagridis is known to infect humans (37). Recent studies have identified six novel avian genotypes: goose genotypes I and II, the duck genotype, two unnamed genotypes in Canada geese (Branta canadensis) (9, 39), and the Eurasian woodcock (Scolopax rusticola) genotype (30). In addition to the host-specific avian genotypes identified, C. hominis and C. parvum were also identified in Canada geese (39). However, this was thought to be due to mechanical transmission (8, 39).

These studies indicate that the extent of genetic diversity among avian-derived Cryptosporidium isolates is greater than previously thought and that this warrants further investigation. The high prevalence of Cryptosporidium in domesticated, caged, and wild birds potentially represents a risk to humans (e.g., pet owners and poultry farmers) and other mammals that become infected with the parasite. The purpose of this study was to examine the prevalence and host range of Cryptosporidium species and genotypes among caged, domesticated, and wild birds and to determine if they are potential reservoirs of human-infectious species of Cryptosporidium.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fecal sample collection and DNA extraction.
A total of 430 fresh fecal samples of various avian host species were randomly collected from March 2004 to December 2004 at selected commercial aviaries, poultry farms, hatcheries, wildlife parks, and the Perth Zoo. All samples were collected into individual 250-ml fecal collection pots and stored at 4°C until required. An additional 11 avian-derived fecal specimens from the Czech Republic that were positive by microscopy for Cryptosporidium were provided by Ivan Pavlasek (Table 1). Fecal samples were processed within 48 h, and total DNA was extracted using a QIAmp DNA Stool Kit (QIAGEN, Hilden, Germany) with minor modifications to the manufacturer's protocol. Briefly, samples were subjected to five cycles of freeze-thaw (following freezing in liquid nitrogen, samples were thawed at 80°C) followed by 10 min of boiling to ensure lysis of the thick-walled Cryptosporidium oocyst and release of DNA. The final elution volume was adjusted to 50 µl of AE buffer (QIAGEN) from the kit manufacturer's recommended volume of 200 µl of AE buffer in order to increase DNA concentration. Morphometric analysis was conducted on positive samples using an Optimus Image Analysis Package, version 5.2, at a magnification of x1,000.

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TABLE 1. Cryptosporidium species and genotypes in avian hosts identified in the present study

PCR amplification and sequence analysis at the 18S rRNA locus.
All 430 fecal samples were screened for the presence of Cryptosporidium at the 18S rRNA locus using a two-step nested PCR as previously described (30). The amplified DNA fragments from the secondary PCR product were separated by gel electrophoresis and purified using the freeze-squeeze method. Briefly, excised gel fragments were placed in a 1.5-ml Eppendorf tube, frozen immediately for a minimum of 2 h, and then squeezed between sheets of parafilm that had been folded twice. The liquid which separated from the gel matrix was extracted with a pipette and collected into a new 1.5-ml Eppendorf tube. The volume of each extraction was determined, and DNA was precipitated by adding 1/10 of the extracted volume of 3 M sodium acetate and 2 volumes of the extracted volume of 70% ethanol. The samples were then centrifuged at 20,000 x g for 5 min, and the supernatant was discarded. The pellet was vacuum dried and redissolved in 15 µl of distilled water. Qualitative and quantitative determination of the eluted DNA was carried out by agarose gel electrophoresis of 2 µl of the eluted DNA with known amounts of DNA. Purified PCR products were then sequenced using an ABI Prism Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, Calif.) according to the manufacturer's instructions, with the exception that the annealing temperature was raised to 58°C.

Actin gene amplification and sequencing.
PCR amplification of the actin gene locus was performed on DNA samples that were positive for Cryptosporidium at the 18S rDNA locus. A PCR product of 830 bp was amplified by a nested PCR using the forward primer actin AII F1 (5'-ATGCCVGGWRTWATGGTDGGTATG-3') and the reverse primer actin Act6R (5'-GGDGCAACRACYTTRATCTTC-3') in the primary PCR. The PCR mixture consisted of 0.01 to 1.0 ng of template DNA, 12.5 µM (each) forward and reverse primer, a 200 µM concentration of each deoxynucleoside triphosphate (dNTP) (Promega), 1xDNA polymerization buffer (Fisher Biotech), 3.0 mM MgCl₂, and 0.5 U of Tth polymerase (Fisher Biotech) in a total reaction mixture of 25 µl. The reactions were performed in a Perkin Elmer Gene Amp PCR 2400 thermocycler with a preliminary cycle of 94°C for 2 min, 58°C for 1 min, and 72°C for 2 min, followed by 50 PCR cycles of 94°C for 30 s, 58°C for 20 s, and 72°C for 40 s, with a final extension of 72°C for 7 min. For the secondary PCR, a fragment of 818 bp was amplified using 1.0 µl of primary PCR and forward actin AII F2 (5'-GAYGARGCHCARTCVAARAGRGGTAT-3') and reverse actin AII R1 (5'-TTDATYTTCATDGTHGAHGGWGC-3') primers. The conditions for the secondary PCR were identical to the primary PCR. PCR products were purified and sequenced as described above.

Phylogenetic analyses.
Nucleotide sequences were analyzed using SeqEd, version 1.0.3 (Applied Biosystems), and aligned using Clustal W (34). Distance estimation was conducted using TREECON (36), based on evolutionary distances calculated with the Tamura-Nei model and grouped using the neighbor-joining method. The confidence of groupings was assessed by bootstrapping using 1,000 replicates. Parsimony analysis was conducted using MEGA, version 3.1 (12). In constructing the neighbor-joining and maximum parsimony trees, a sequence of Eimeria faurei (GenBank accession no. AF345998) was used as an outgroup for the 18S rDNA analysis, whereas Plasmodium falciparum (GenBank accession no. M19146) was used as the outgroup in the analysis of the actin genes. Sequences of additional Cryptosporidium species and genotypes were obtained from the GenBank database (http://www.ncbi.mln.nih.gov/GenBank/index.html).

Nucleotide sequence accession numbers.
The unique partial 18S rRNA and actin sequences generated as part of this study have been deposited in the GenBank database under accession numbers DQ650339 to DQ650350.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cryptosporidium spp. in feces of avian hosts.
Of the 430 Australian avian-derived fecal specimens examined in the present study, a total of 27 (6.3%) samples were positive for Cryptosporidium at the 18S rRNA locus (Table 1). Sequence and phylogenetic analysis identified C. galli (9 isolates) and three novel genotypes among the Australian isolates: avian genotype I (1 isolate), avian genotype II (12 isolates), and avian genotype III (5 isolates) (Fig. 1). Of the 11 avian-derived Cryptosporidium isolates from the Czech Republic, a total of five species—C. galli (4 isolates), C. baileyi (3 isolates), C. baileyi-like (1 isolate, B4-26), C. andersoni (1 isolate), and C. muris (1 isolate)—and one novel genotype, avian genotype IV (1 isolate, B4-33) were identified. C. meleagridis, C. hominis, and C. parvum were not detected in any of the samples tested.

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FIG. 1. Evolutionary relationships of Cryptosporidium isolates inferred by neighbor-joining analysis of Tamura-Nei distances calculated from pairwise comparison of 18S rRNA sequences. Percentage bootstrap support (>60%) from 1,000 pseudoreplicates (analyzed by neighbor-joining and parsimony) is indicated at the left of the supported node. nr, node not recovered by method. Origins of the avian genotypes identified are in brackets (WA, Western Australia; CzR, Czech Republic). Accession numbers are given in parentheses.

Morphometric analysis.
Oocysts were present in low numbers in all the samples analyzed, and therefore only limited morphometric analysis was conducted. Oocysts from avian genotype II measured 6.5 by 6.6 µm, which is larger than the oocysts of its closest relative, C. baileyi (6.4 by 4.8 µm) (38). Oocysts from avian genotype III measured 7.5 by 6.0 µm, which is larger than the mean oocyst size of C. serpentis (5.94 by 5.11 µm) but smaller than the oocyst size of the Eurasian woodcock-derived oocysts (8.25 by 6.3 µm) (30). Oocysts from avian genotype IV were similar in size to C. galli and measured 8.25 by 6.3 µm.

Genetic relationships among avian-derived Cryptosporidium spp. and genotypes.
Distance- and parsimony-based phylogenetic analysis revealed that avian genotypes I, III, and IV were genetically distinct from any previously described species or genotypes of Cryptosporidium, whereas avian genotype II isolates were identical to a novel Cryptosporidium genotype recently identified in ostriches (18). Although avian genotype II was first identified in ostriches, it appears to have a broad host range (Table 1), and therefore we have retained the name avian genotype II rather than the ostrich genotype. Sequence and distance analysis at the 18S rRNA gene locus indicated that although avian genotype I and avian genotype II were most closely related to C. baileyi, they were genetically distinct (99.5% and 97.4% similarity, respectively, to C. baileyi) and exhibited 98.8% similarity to each other (Table 2 and Fig. 1 and 2). At the actin gene locus, avian genotype I and avian genotype II exhibited only 95.7% and 88.3% similarity, respectively, to C. baileyi. Parsimony analysis produced essentially the same trees as distance-based methods (data not shown).

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TABLE 2. Percent similarity of the Cryptosporidium spp. to their closest relative and the range of accepted Cryptosporidium spp. at the 18S rRNA and actin gene loci

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FIG. 2. Evolutionary relationships of Cryptosporidium isolates inferred by neighbor-joining analysis of Tamura-Nei distances calculated from pairwise comparison of actin DNA sequences. Percentage bootstrap support (>60%) from 1,000 pseudoreplicates (analyzed by neighbor-joining and parsimony) is indicated at the left of the supported node. nr, node not recovered by method. Accession numbers are given in parentheses.

Avian genotype III formed a distinct group, which clustered with the Eurasian woodcock genotype and the rest of the gastric Cryptosporidium parasites (Table 2 and Fig. 1 and 2). The avian genotype III exhibited 98.7% genetic similarity to the Eurasian woodcock and 98.3% genetic similarity to C. serpentis and shared 95.7 to 97.5% similarity with the other gastric parasites (C. galli, C. muris, and C. andersoni) at the 18S rRNA locus. At the actin gene locus, avian genotype III exhibited 98.5%, 94.6%, and 96.6% genetic similarity to the Eurasian woodcock, C. serpentis, and C. galli, respectively (Table 2 and Fig. 1 and 2). Parsimony analysis produced essentially the same tree topologies as distance analysis (data not shown).

Avian genotype IV was identified in a Japanese white-eye (Zosterops japonica) from the Czech Republic and exhibited 96.5% similarity to its closest relative, C. galli, at the 18S rRNA locus, but we were unable to obtain a sequence at the actin locus.

Two novel avian genotypes (geese 3b and 7) identified in a previous study (9) were not included in the phylogenetic analysis in the present study because the partial sequence of the 18S rRNA available for comparison only overlapped for approximately 230 bp of the 375-bp rRNA sequence in the present study. However, partial alignments of the avian genotypes I, II, II, and IV with the novel sequences identified from geese 3b and 7 revealed them to be distinct.

Clinical signs.
No clinical signs, such as diarrhea, dyspnoea, coughing, or sneezing were detected in the Australian Cryptosporidium-positive avian hosts examined in the present study. The Czech Republic Cryptosporidium-positive avian hosts exhibited diarrhea and anorexia. Microscopic analysis detected only Cryptosporidium oocysts; no other pathogenic bacteria were observed.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of this study indicate that the extent of genetic diversity among avian hosts is greater than previously thought as four species (C. galli, C. baileyi, C. muris, and C. andersoni), avian genotype II (similar to a novel genotype from an ostrich), and three novel genotypes (avian genotypes I, III, and IV) were identified among the Australian and Czech avian isolates analyzed. Avian genotype II, C. galli, and avian genotype III were the most common Cryptosporidium spp. detected in the Australian avian hosts; they had prevalence rates of 2.8%, 2.1%, and 1.2%, respectively. Avian genotype IV was detected in only one isolate from the Czech Republic and not in the Australian avian hosts. The overall prevalence rate of 6.3% for Cryptosporidium spp. in the Australian avian hosts was low compared to previous studies which have reported prevalences ranging from 6 to 100% in farm-raised chickens (2, 7, 22, 27, 33), 20 to 60% in ducks and geese (9, 11, 33, 39), and 22 to 100% in gulls (23, 33). All the Australian fecal samples were obtained from aviaries and commercial poultry farms where strict sanitation procedures were in place including daily cleaning of cages and aviaries (e.g., changing paper trays and washing of the aviaries and water and food containers), which may account for the lower prevalences reported in the present study.

The host range for avian genotype II included cockatiels, cockatoos, a parrot, a galah, and a sun conure (Table 1). The host range for avian genotype III was similar and included a sun conure, a galah, and cockatiels. Both avian genotypes I and IV were identified in single avian hosts, a red factor canary (Serinus canaria) and a Japanese white-eye (Zosterops japonica), respectively. Limited morphometric analysis (<10 oocysts per genotype) suggested that avian genotypes II and III are morphologically distinct; however, larger numbers of oocysts from each genotype (50) would need to be analyzed in order to produce statistically meaningful results.

C. galli was identified in nine Australian avian-derived isolates (a prevalence of 2.1%) and in three isolates from the Czech Republic, extending the host range for this species, as it has not been previously reported in turquoise parrots, Cuban flamingos, red-cowled cardinals, and rhinocerous hornbills (Table 1).

C. baileyi has been previously identified in chickens, turkeys, ducks, cockatiels, and geese (1, 9, 16, 19) and in the present study was identified in four avian hosts including a chicken, a crane, a weaver, and a bulbul (Table 1), extending the host range of this species. Interestingly, C. meleagridis was not detected in the present study in Australian birds even though C. meleagridis and C. baileyi have previously been thought to be the two most common avian species of Cryptosporidium (4, 6, 21, 33). C. hominis and C. parvum were also not detected, indicating that caged, domesticated, and wild birds are not normal hosts for these species and are unlikely to be sources of human infection.

C. muris (isolate B3-15) and C. andersoni (isolate B4-25) were identified in a tawny frogmouth and a quail-crested wood partridge, respectively. This is the first time to the best of our knowledge that these species have been reported in avian hosts, but whether the presence of the oocysts in the feces was due to mechanical transport or an actual infection remains to be determined.

Avian Cryptosporidium have been detected in over 30 species of birds belonging to six different orders (Anseriformes, Charadriiformes, Columbiformes, Galliformes, Passeriformes, Psittaciformes, and Struthioniformes) (21, 33). From the diverse range of avian host species (Anseriformes, Caprimulgiformes, Columbiformes, Coraciiformes, Galliformes, Passeriformes, Psittaciformes, Stringiformes, and Struthioniformes) examined in this study, avian genotypes II and III were identified only in Psittaciformes, whereas C. galli was identified in a wider range of birds (Psittaciformes, Passeriformes, and Galliformes). However, it would appear that avian genotype II is also capable of infecting Struthioniformes as this genotype has previously been identified in ostriches (18). In the present study, fecal samples from ostriches were also examined; however, avian genotype II was not identified in any of these samples. Further research is required to determine the full host range of avian genotypes II and III, and it is likely that as a broader range of bird species is examined, more avian Cryptosporidium spp. will be detected.

The results of this study indicate that Cryptosporidium is able to infect a large range of avian species and that, potentially, there are more avian Cryptosporidium species yet to be identified. It also suggests that pet birds may play an important role in the epidemiology of Cryptosporidium. The potential of these newly identified genotypes to cause disease in birds or humans is unknown, and further studies are required to understand the extent of host adaptation for avian-derived species of Cryptosporidium and the public health implications, if any, of the novel species identified in this study.

 
* Corresponding author. Mailing address: Divison of Veterinary and Biomedical Sciences, Murdoch University, South Street, Murdoch, WA 6150, Australia. Phone: 61 8 9360 2495. Fax: 61 8 9310 4144. E-mail: J.Ng{at}murdoch.edu.au.

Published ahead of print on 6 October 2006.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, December 2006, p. 7548-7553, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01352-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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