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.

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.

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.  [26,27]. Results were considered statistically significant when < 0.05. Odds-ratios (OR) with 95% confidence intervals based on likelihood ratio statistics were reported.

Results and Discussion
Of 311 parrots, ten (3.22%) were positive for Cryptosporidium infection by ELISA (  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 ( < 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). 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   [19] 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.

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.