Prevalence and Genotyping of Cryptosporidium Infection in Pet Parrots in North China.

Cryptosporidiosis is a worldwide zoonosis caused by Cryptosporidium spp., sometimes leading to severe diarrhea in humans and animals. In the present study, 311 parrots, belonging to four species, namely, Budgerigars (Melopsittacus undulatus), Lovebirds (Agapornis sp.), Alexandrine parakeets (Psittacula eupatria), and Cockatiel (Nymphicus hollandicus), from Beijing and Weifang cities, were examined for Cryptosporidium spp. infection. Blood samples of each bird were examined using enzyme linked immunosorbent assay (ELISA) and fecal samples were examined by Sheather's sugar flotation technique. Prevalence of Cryptosporidium infection were 3.22% (10/311) and 0.64% (2/311) by ELISA and Sheather's sugar flotation technique, respectively. Seroprevalence of Cryptosporidium infection in different breeds varied from 0 to 15.39%. Sequencing analysis showed that both positive samples from fecal samples belonged to Cryptosporidium avian genotype V. This is the first report of Cryptosporidium avian genotype V in Budgerigars. The results of the present study provided foundation-data for prevention and control of cryptosporidiosis in pet birds in China.

1. Introduction

Cryptosporidiosis, caused by the enteric parasite pathogens Cryptosporidium spp., can lead to diarrheal illness in humans and animals including birds [1-3]. Since Tyzzer [4] firstly observed the Cryptosporidium infection in birds, this pathogen has been detected in more than 30 avian species worldwide [5]. Recent molecular epidemiologic studies identified a number of genetically distinct avian genotypes, including the Eurasian woodcock genotype, the black duck genotype, the goose genotypes (I–IV), and avian genotypes (I–V) [6-12].

Infection with Cryptosporidium species such as C. meleagridis, C. baileyi, C. galli, C. parvum, avian genotype II, avian genotype III, and avian genotype V in parrots has been widely reported in Japan, Brazil, and Australia [6, 9, 10, 13–17]. Cryptosporidium infection in birds has also been reported in China, and these reports are listed in Table 1.

Table 1

Prevalence of Cryptosporidium infection in birds in China in previous studies.

Geographic origin	Host species	Scientific name	Cryptosporidium spp.	Prevalence (%)	Reference
Qinghai Lake	Ruddy Shelduck	Tadorna ferruginea	C. baileyi	3.38 (5/148)	[20]
Zhengzhou city	Black-billed magpie	Pica pica	C. baileyi	100 (1/1)	[19]
Zhengzhou city	Bohemian waxwing	Bombycilla garrulus	C. Meleagridis, C. galli	55.6 (5/9)	[19]
Zhengzhou city	Cockatiel	Nymphicus hollandicus	Avian genotype V, avian genotype III	20.5 (8/39)	[19]
Zhengzhou city	Common myna	Acridotheres tristis	C. baileyi	11.1 (4/36)	[19]
Zhengzhou city	Crested Lark	Galerida cristata	C. baileyi	11.1 (1/9)	[19]
Zhengzhou city	Fan-tailed pigeon	Columba livia	C. meleagridis	4.8 (1/21)	[19]
Zhengzhou city	Gouldian finch	Chloebia gouldiae	C. baileyi	14.3 (1/7)	[19]
Zhengzhou city	Red-billed blue magpie	Urocissa erythrorhyncha	Avian genotype III	100 (1/1)	[19]
Zhengzhou city	Red-billed leiothrix	Leiothrix lutea	C. baileyi	11.4 (5/45)	[19]
Zhengzhou city	Rufous turtle dove	Streptopelia orientalis	C. meleagridis	50 (1/2)	[19]
Zhengzhou city	Silver-eared Mesia	Leiothrix argentauris	C. galli	14.3 (1/7)	[19]
Zhengzhou city	White Java sparrow	Padda oryzivora	C. baileyi	16 (4/25)	[19]
Zhengzhou city	Zebra finch	Taeniopygia guttata	C. baileyi	5 (2/40)	[19]
Zhengzhou city	Ostriches	Struthio camelus	C. muris, C. baileyi	10.2 (31/311)	[21]
Henan province	Pekin ducks	Anas platyrhynchos	C. baileyi	16.3 (92/564)	[22]
Henan province	Chickens	Gallus domestiaus	C. meleagridis, C. baileyi	8.9 (179/2015)	[22]
Zhengzhou city	Ostriches	Struthio camelus	C. baileyi	11.7 (53/452)	[23]
Henan province	Quails	Coturnix coturnix japonica	C. baileyi, C. meleagridis	13.1 (239/1818)	[24]

In China, parrots have been raised and kept over a long-term history for companionship and entertainment [18]. However, except a study on detection of avian genotypes III and avian genotype V in Cockatiel (Nymphicus hollandicus) in Henan province [19], no such information on Cryptosporidium prevalence and genetic diversity in other species of parrots is available in China. The aims of the present study were to examine the prevalence of Cryptosporidium infection and identify Cryptosporidium spp. in Budgerigars (Melopsittacus undulatus), Lovebirds (Agapornis sp.), Alexandrine parakeets (Psittacula eupatria), and Cockatiel in north China.

2. Materials and Methods

2.1. Ethic Statement

Data regarding species, geographic origin, age, and gender were obtained from local veterinary practitioner. All birds were handled in strict accordance with the Good Animal Practice requirements of the Animal Ethics Procedures and Guidelines of the People's Republic of China. This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (approval number LVRIAEC2012-010).

2.2. Investigated Sites and Sampling

The survey was carried out in Beijing and Weifang cities (two main locations of parrots' production), northern China. The two cities belong to north temperate and monsoonal climate with an average annual temperature of about 13.0°C. A total of 311 samples were collected from Budgerigars, Lovebirds, Cockatiel, and Alexandrine parakeets from pet shops from March to June 2013. The blood samples were collected from wing vein of each parrot by using a 2–5 mL vacuum blood collection tube (without an anticoagulant), and then blood samples were sent to the laboratory and separated by centrifugation at 3,000 g for 10 min to obtain serum samples. Meanwhile, cloacal swabs samples were collected by using an aseptic cotton and then filtered via a 0.3 mm wire mesh, and the filtrate was transferred into a 1.5 mL tube, followed by centrifuged at room temperature at 1000 g for 10 min. After discarding the supernatant, the concentrated fecal specimens were used for further analysis.

2.3. Examination of Cryptosporidium Infection

All serum samples were examined for the presence of Cryptosporidium antibodies by enzyme linked immunosorbent assay (ELISA) (Nuoyuan Co., Ltd., Shanghai, China) according to the manufacturer's instruction. Fecal samples of each parrot were examined using Sheather's sugar flotation technique. Positive fecal samples were used to molecularly determine Cryptosporidium spp. Genomic DNA was extracted using the Stool DNA kit (OMEGA, USA) as instructed by the manufacturer. The nested-PCR based on the small subunit (SSU) rRNA gene was performed as previously described [25]. The second PCR products were sequenced by Shanghai Sangon Company. The sequence obtained was deposited in GenBank with the accession number of KM267556.

2.4. Phylogenetic Relationships of Cryptosporidium spp

The obtained Cryptosporidium nucleotide sequence was aligned with corresponding sequences from the GenBank database using the BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and ClustalX 1.83 (http://www.clustal.org/). A phylogenetic tree was constructed by the Neighbor-Joining (NJ) analysis of the SSU rRNA sequences in Mega 5.0 (http://www.megasoftware.net/) with Kimura 2-parameter model and 1000 replicates.

2.5. Statistical Analysis

Differences in the prevalence of Cryptosporidium infection in parrots among different locations, ages, genders, and species were analyzed using SAS software (version 9.1, SAS Institute, Inc., Cary, NC) [26, 27]. Results were considered statistically significant when P < 0.05. Odds-ratios (OR) with 95% confidence intervals based on likelihood ratio statistics were reported.

3. Results and Discussion

Of 311 parrots, ten (3.22%) were positive for Cryptosporidium infection by ELISA (Table 2), with three (two female parrots in June, one male parrots in March) collected from Beijing and seven (two female parrots, five male parrots) collected from Weifang. Seroprevalence of Cryptosporidium infection in different breeds varied from 0 to 15.39%, and the difference was statistically significant (P < 0.05) (Table 2). However, only two (0.64%) Cryptosporidium-positive fecal samples were detected by Sheather's sugar flotation technique, with one from a female Cockatiel in Beijing in June and the other in male Budgerigars in Weifang in March. Sequence and phylogenetic analysis indicated that only one Cryptosporidium genotype (avian genotype V) was identified from the two fecal-positive samples (Figure 1).

Table 2

Seroprevalence of Cryptosporidium infection in parrots in different regions, sexes, species, ages, and seasons by enzyme linked immunosorbent assay (ELISA) in this study.

Variable	Category	Number of tested samples	Number of positive samples	Prevalence (%) (95% CI)	P value	OR (95% CI)
Region	Beijing	158	3	1.90 (0.00–4.03)	0.18	Reference
	Weifang	153	7	4.58 (1.26–7.89)		2.48 (0.63–9.76)
Sex	Male	163	6	3.68 (0.79–6.57)	0.63	Reference
	Female	148	4	2.70 (0.09–5.32)		0.73 (0.20–2.63)
Species	Budgerigar (Melopsittacus undulatus)	202	4	1.98 (0.06–3.90)	0.0005	Reference
	Alexandrine parakeets (Psittacula  eupatria)	61	0	0.00 (—)		—
	Lovebirds (Agapornis sp.)	26	4	15.39 (1.52–29.25)		9.00 (2.10–38.53)
	Cockatiel (Nymphicus hollandicus)	22	2	9.09 (0.00–21.10)		4.95 (0.85–28.73)
Age	≤5 months	105	4	3.81 (0.15–7.47)	0.63	Reference
	6–12 months	100	4	4.00 (0.16–7.84)		1.05 (0.26–4.33)
	13–18 months	106	2	1.89 (0.00–4.48)		0.49 (0.09–2.71)
Season	Spring	139	5	3.60 (0.50–6.69)	0.73	Reference
	Summer	172	5	2.91 (0.40–5.42)		0.80 (0.23–2.83)
Total		311	10	3.22 (1.26–5.18)

Figure 1

Phylogenetic analyses of Cryptosporidium spp. using Neighbor-Joining (NJ) method based on sequences of the small subunit ribosomal RNA (SSU rRNA) gene. The Cryptosporidium isolate identified in the present study is underlined.

In the present study, the overall prevalence of Cryptosporidium infection tested by Sheather's sugar flotation technique was 0.64%, which is lower than that of Japanese Quail (Coturnix coturnix japonica) (13.1%) [24], chickens (8.9%), Pekin ducks (Anas platyrhynchos) (16.3%), and Ostriches (Struthio camelus) (10.2%) in Zhengzhou of Henan province [21, 22], Ruddy Shelduck (Tadorna ferruginea) (3.38%) in Qinghai Lake [20], birds in Brazil (6.6% and 4.84%) [15, 16], and avian in Australia (6.28%) [10], but higher than that in birds in Taiwan (0%) [28]. Low oocyst counts in fecal samples and the sampling time out of the oocysts shedding period may contribute to the low detecting rates of the parasite by microscopy [29]. In general, because of test methods, sample sizes, and geoecological conditions, the actual discrepancy is difficult to explain in the prevalence of Cryptosporidium among different studies [30]. In this investigation, we detected higher seroprevalence (10/311, 3.22%) of Cryptosporidium infection in parrots compared with Sheather's sugar flotation technique. This is because ELISA usually has better sensitivity for the detection of antibodies against Cryptosporidium [31]. Moreover, parrots which were positive for Cryptosporidium oocysts in fecal samples were also positive for indirect ELISA.

Seven Cryptosporidium species/genotypes, namely, avian genotype II, avian genotype III, avian genotype V, C. meleagridis, C. baileyi, C. galli, and C. parvum, have been identified in parrots in previous studies (Table 3). However, in the present study, only one Cryptosporidium genotype was detected and identified in Budgerigar. A BLAST similarity search indicated that the obtained sequences of SSU rRNA gene were 100% identical to the Cryptosporidium avian genotype V (GenBank accession numbers: HM116381 and AB471647), which was recently reported in Cockatiel in Zhengzhou city of China [19] and Japan [6], respectively. However, other six Cryptosporidium species/genotypes were not detected in parrots in this study, which may be related to the small sample size. Further studies are needed to expand the sample size to detect the Cryptosporidium species/genotypes in parrots in China, which could contribute to estimating the zoonotic potential of Cryptosporidium from parrots.

Table 3

Occurrence of Cryptosporidium spp. performed with 18S rDNA in parrots in the world in previous studies (available data).

Geographic origin	Host species	Scientific name	Cryptosporidium spp.	Reference
Japan	Cockatiel	Nymphicus hollandicus	C. meleagridis, avian genotype III, avian genotype V	[6]
Japan	Cockatiel	Nymphicus hollandicus	C. meleagridis and C. baileyi	[13]
Japan	Peach-faced lovebird	Agapornis roseicollis	Avian genotype III	[14]
Australia	Indian ring-necked parrot	Psittacula krameri	C. meleagridis	[9]
Australia	Cockatiel	Nymphicus hollandicus	Avian genotype II, avian genotype III	[10]
Australia	Major Mitchell cockatoo	Cacatua leadbeateri	Avian genotype II	[10]
Australia	Eclectus	Eclectus roratus	Avian genotype II	[10]
Australia	Galah	Eolophus roseicapilla	Avian genotype II, avian genotype III	[10]
Australia	Turquoise parrots	Neophema pulchella	C. galli	[10]
Australia	Sun conure	Aratinga solstitialis	Avian genotype II, avian genotype III	[10]
Australia	Princess parrot	Polytelis alexandrae	Avian genotype II	[10]
Australia	Alexandrine	Psittacula eupatria	Avian genotype II	[10]
Brazil	Cockatiel	Nymphicus hollandicus	C. galli, C. parvum, avian genotype III	[15]
Brazil	Peach-faced lovebird	Agapornis roseicollis	Avian genotype III	[15]
Brazil	white-eyed parakeet	Aratinga leucophthalma	Avian Genotype II	[16]
Brazil	Cockatiel	Nymphicus hollandicus	C. galli	[17]
China	Cockatiel	Nymphicus hollandicus	Avian genotype III, avian genotype V	[19]

4. Conclusion

The results of the present study revealed the existence of avian genotype V infection in Budgerigars in North China, which provided foundation-data for prevention and control of cryptosporidiosis in pet birds in China.