# Prevalence and Genotyping ofCryptosporidium Infection in Pet Parrots in North China

Authors: Xiao-Xuan Zhang; Nian-Zhang Zhang; Guang-Hui Zhao; Quan Zhao; Xing-Quan Zhu
Journal: BioMed Research International
 (2015)

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## Abstract

Cryptosporidiosis is a worldwide zoonosis caused byCryptosporidium 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.


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## Body

## 1. Introduction

Cryptosporidiosis, caused by the enteric parasite pathogensCryptosporidiumspp., can lead to diarrheal illness in humans and animals including birds [1–3]. Since Tyzzer [4] firstly observed theCryptosporidium 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 withCryptosporidium species such asC. 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 ofCryptosporidium 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 onCryptosporidium prevalence and genetic diversity in other species of parrots is available in China. The aims of the present study were to examine the prevalence ofCryptosporidium infection and identifyCryptosporidium spp. in Budgerigars (Melopsittacus undulatus), Lovebirds (Agapornissp.), 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 ofCryptosporidium Infection

All serum samples were examined for the presence ofCryptosporidium 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 determineCryptosporidium 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 ofCryptosporidium spp.

The obtainedCryptosporidium 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 ofCryptosporidium 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.


## 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 ofCryptosporidium Infection

All serum samples were examined for the presence ofCryptosporidium 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 determineCryptosporidium 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 ofCryptosporidium spp.

The obtainedCryptosporidium 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 ofCryptosporidium 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 forCryptosporidium 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 ofCryptosporidiuminfection 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 oneCryptosporidiumgenotype (avian genotype V) was identified from the two fecal-positive samples (Figure 1).Table 2
Seroprevalence ofCryptosporidium 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 (Agapornissp.)
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 ofCryptosporidium spp. using Neighbor-Joining (NJ) method based on sequences of the small subunit ribosomal RNA (SSU rRNA) gene. TheCryptosporidium isolate identified in the present study is underlined.In the present study, the overall prevalence ofCryptosporidiuminfection 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 ofCryptosporidium among different studies [30]. In this investigation, we detected higher seroprevalence (10/311, 3.22%) ofCryptosporidiuminfection in parrots compared with Sheather’s sugar flotation technique. This is because ELISA usually has better sensitivity for the detection of antibodies againstCryptosporidium [31]. Moreover, parrots which were positive forCryptosporidiumoocysts in fecal samples were also positive for indirect ELISA.SevenCryptosporidium species/genotypes, namely, avian genotype II, avian genotype III, avian genotype V,C. meleagridis,C. baileyi,C. galli, andC. parvum, have been identified in parrots in previous studies (Table 3). However, in the present study, only oneCryptosporidium genotype was detected and identified in Budgerigar. A BLAST similarity search indicated that the obtained sequences of SSU rRNA gene were 100% identical to theCryptosporidium 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 sixCryptosporidium 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 theCryptosporidium species/genotypes in parrots in China, which could contribute to estimating the zoonotic potential ofCryptosporidiumfrom parrots.Table 3
Occurrence ofCryptosporidium 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.



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