# Prevalence and Genetic Characterization ofCryptosporidium Infection in Java Sparrows (Lonchura oryzivora) in Northern China

Authors: Qiu-Xia Yao; Xiao-Xuan Zhang; Kai Chen; Jian-Gang Ma; Wen-Bin Zheng; Xiao-Qin Xu; Xing-Quan Zhu
Journal: BioMed Research International
 (2017)

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## Abstract

Cryptosporidiosis is a cosmopolitan parasitosis that affects a wide range of hosts including birds. As information concerningCryptosporidium in birds is limited, the present study examined the prevalence and genotypes ofCryptosporidium in Java sparrows in Beijing and Shangqiu, northern China. Three hundred and fifty fecal samples were collected from Java sparrows (Lonchura oryzivora, 225 white Java sparrows and 125 gray Java sparrows) in Beijing and Shangqiu in October 2015, and the samples were examined by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene. The overallCryptosporidium prevalence is 13.42% (47/350), with 16.44% (37/225) in white Java sparrows and 8.00% (10/125) in gray Java sparrows.Cryptosporidium prevalence was 9.82% (16/163) in Java sparrows from Beijing and 16.58% (31/187) in Java sparrows from Shangqiu. The prevalence ofCryptosporidium in females and males was 40.63% (26/64) and 7.34% (21/286), respectively. TheCryptosporidium prevalence in Java sparrows of different ages varied from 10.47% to 16.33%. Sequence analysis of the SSU rRNA gene revealed that all the samples representedC. baileyi. This is the first report ofCryptosporidium in gray Java sparrows in China, which extend the host range forC. baileyi. These results provide baseline information for further studies of molecular epidemiology and control ofCryptosporidium infection in poultry in China.


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## Body

## 1. Introduction

Enteric parasiteCryptosporidium is one of the most important causative agents of gastrointestinal illnesses in humans and animals [1–3]. Food and water contaminated byCryptosporidium oocysts are the most important resources for human infection [3–5]. Moreover, it can be transmitted to humans by direct contact with infected animals [3, 6]. Human infection withCryptosporidium may show symptoms of diarrheal illness and some other severe diseases.Cryptosporidium infection in birds may present symptoms of respiratory disease, digestive symptoms, and renal disease [7, 8].Cryptosporidium infection in birds was firstly recorded in 1929. So far, over 30 avian species have been reported as the hosts ofCryptosporidium in the world [7]. Respiratory symptoms frequently occur in birds, and this infection leads to higher morbidity and mortality in birds, especially in broiler chickens [7]. Recently, more than 17Cryptosporidium species/genotypes (C. galli,C. baileyi,C. meleagridis,C. hominis,C. parvum,C. muris,Cryptosporidium avian I–V, goose genotypes I–IV, duck genotype, and Eurasian woodcock genotype) have been recorded in birds worldwide [7, 9–13]. Of these,C. hominis,C. parvum, andC. meleagridis are well-known causative agents of human infection [12].In view of such severe situations, a number of studies concerningCryptosporidium prevalence and genotypes in birds have been conducted worldwide. In China, a few studies onCryptosporidium infection in birds have been reported in Henan [12], Qinghai [14], and Zhejiang [15] provinces. A preliminary survey indicated thatCryptosporidium could infect white Java sparrows (Lonchura oryzivora) [12], but the number of sampled birds is too small (n=25), which may not reflect the actual prevalence ofCryptosporidium in this bird. Also, no information is available about the prevalence ofCryptosporidium in gray Java sparrows in China. Therefore, we investigated theCryptosporidium prevalence in Java sparrows (Lonchura oryzivora) in China and assessed its associated risk factors.


## 2. Materials and Methods

### 2.1. The Investigated Sites

The survey was conducted in Beijing and Shangqiu cities (two main locations of birds’ production), northern China. Beijing city (39°26′–41°03′N, 115°25′–117°30′E) lies to the south of the Yanshan Mountains with an average altitude of 43.5 m, annual precipitation of 626 mm, and average annual temperature of 12.6°C. Shangqiu city (114°82′–116°45′E, 33°98′–34°80′N) is located in Henan province and has a subhumid warm temperate continental monsoonal climate. The altitude of Shangqiu city ranges from 30 m to 70 m, and the average annual temperature is 14.2°C.


### 2.2. Collection and Preparation of Samples

A total of 350 fecal samples were collected randomly from 225 white Java sparrows (Lonchura oryzivora) and 125 gray Java sparrows (Lonchura oryzivora), of which 163 were obtained from Beijing and 187 were obtained from Shangqiu from pet shops in 2015. Fecal samples were collected by using aseptic cotton and then filtered (0.3 mm wire mesh), and the filtrate was transferred into a 1.5 mL tube. All the samples were stored at 4°C until used for further analysis. Genomic DNA was extracted from feces using a Stool DNA kit (OMEGA, USA) according to the manufacturer’s instructions. The DNA samples were stored at −20°C until used. Information about species, origin, gender, and age of Java sparrows was obtained and listed in Table 1.Table 1
Factors associated with prevalence ofCryptosporidium in Java sparrows in Beijing and Shangqiu, northern China.
Factor
Category
Number positive
Number tested
Prevalence (%) (95% CI)
OR (95% CI)
P value
Region
Beijing
16
163
9.82 (5.26–14.38)
Reference
0.06
Shangqiu
31
187
16.58 (11.25–21.91)
0.55 (0.29–1.04)
Species
White Java sparrow
37
225
16.44 (11.60–21.28)
Reference
0.03
Gray Java sparrow
10
125
8.00 (3.24–12.76)
2.26 (1.08–4.73)
Sex
Female
26
64
40.63 (28.60–52.66)
Reference
<0.01
Male
21
286
7.34 (2.22–12.46)
8.63 (4.43–16.84)
Age
3~5 months
11
92
11.95 (5.33–18.57)
Reference
0.38
6~8 months
25
153
16.33 (10.47–22.19)
0.70 (0.49–2.82)
>8 months
11
105
10.47 (4.61–16.33)
Total
47
350
13.42 (9.85–16.99)


### 2.3. PCR Amplification

Cryptosporidium species/genotypes were determined by nested PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene using primers F1 (5′-CCCATTTCCTTCGAAACAGGA-3′) and R1 (5′-TTCTAGAGCTAATACATGCG-3′) for primary amplification and F2 (5′-AAGGAGTAAGGAACAACCTCCA-3′) and R2 (5′-GGAAGGGTTGTATTTATTAGATAAAG-3′) for secondary amplification [16, 17]. PCR reaction (25 μL) was composed of 1x PCR buffer (Mg2+-free), 2 mM MgCl2, 200 μM of each deoxyribonucleoside triphosphate (dNTP), 0.4 μM of each primer, 0.2 U of HotStartTaq DNA polymerase (Takara, Dalian, China), and 2 μL of DNA template. The cycling conditions were 5 min at 95°C for initial denaturation and then 35 cycles of 45 s at 94°C, 45 s at 55°C, and 1 min at 72°C, followed by final extension at 72°C for 10 min. Both positive and negative controls were included in each test. PCR products were observed under UV light after electrophoresis in 1.5% agarose gel containing GoldView™ (Solarbio, Beijing, China).


### 2.4. Sequencing Analyses

The positive PCR products of Java sparrows were sequenced by GenScript Company (Nanjing, China).Cryptosporidium species were determined by alignment with known reference sequences available in GenBank using BLAST (https://www.ncbi.nlm.nih.gov/BLAST/). Representative nucleotide sequences were deposited in GenBank under accession numbers KY034418-KY034427 and KY962159.


### 2.5. Statistical Analysis

The variation inCryptosporidium prevalence (y) of Java sparrows of different geographical location (x1), age (x2), species (x3), and gender (x4) was analyzed by χ2 test using SPSS 19.0. In the multivariable regression analysis, each of these variables was included in the binary logit model as an independent variable. The best model was judged by Fisher’s scoring algorithm. All tests were two-sided, and values of probability (P) < 0.05 were considered statistically significant. Odds ratios (ORs) and their 95% confidence intervals (95% CIs) were estimated to explore the strength of the association betweenCryptosporidium positivity and the conditions tested.


## 2.1. The Investigated Sites

The survey was conducted in Beijing and Shangqiu cities (two main locations of birds’ production), northern China. Beijing city (39°26′–41°03′N, 115°25′–117°30′E) lies to the south of the Yanshan Mountains with an average altitude of 43.5 m, annual precipitation of 626 mm, and average annual temperature of 12.6°C. Shangqiu city (114°82′–116°45′E, 33°98′–34°80′N) is located in Henan province and has a subhumid warm temperate continental monsoonal climate. The altitude of Shangqiu city ranges from 30 m to 70 m, and the average annual temperature is 14.2°C.


## 2.2. Collection and Preparation of Samples

A total of 350 fecal samples were collected randomly from 225 white Java sparrows (Lonchura oryzivora) and 125 gray Java sparrows (Lonchura oryzivora), of which 163 were obtained from Beijing and 187 were obtained from Shangqiu from pet shops in 2015. Fecal samples were collected by using aseptic cotton and then filtered (0.3 mm wire mesh), and the filtrate was transferred into a 1.5 mL tube. All the samples were stored at 4°C until used for further analysis. Genomic DNA was extracted from feces using a Stool DNA kit (OMEGA, USA) according to the manufacturer’s instructions. The DNA samples were stored at −20°C until used. Information about species, origin, gender, and age of Java sparrows was obtained and listed in Table 1.Table 1
Factors associated with prevalence ofCryptosporidium in Java sparrows in Beijing and Shangqiu, northern China.
Factor
Category
Number positive
Number tested
Prevalence (%) (95% CI)
OR (95% CI)
P value
Region
Beijing
16
163
9.82 (5.26–14.38)
Reference
0.06
Shangqiu
31
187
16.58 (11.25–21.91)
0.55 (0.29–1.04)
Species
White Java sparrow
37
225
16.44 (11.60–21.28)
Reference
0.03
Gray Java sparrow
10
125
8.00 (3.24–12.76)
2.26 (1.08–4.73)
Sex
Female
26
64
40.63 (28.60–52.66)
Reference
<0.01
Male
21
286
7.34 (2.22–12.46)
8.63 (4.43–16.84)
Age
3~5 months
11
92
11.95 (5.33–18.57)
Reference
0.38
6~8 months
25
153
16.33 (10.47–22.19)
0.70 (0.49–2.82)
>8 months
11
105
10.47 (4.61–16.33)
Total
47
350
13.42 (9.85–16.99)


## 2.3. PCR Amplification

Cryptosporidium species/genotypes were determined by nested PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene using primers F1 (5′-CCCATTTCCTTCGAAACAGGA-3′) and R1 (5′-TTCTAGAGCTAATACATGCG-3′) for primary amplification and F2 (5′-AAGGAGTAAGGAACAACCTCCA-3′) and R2 (5′-GGAAGGGTTGTATTTATTAGATAAAG-3′) for secondary amplification [16, 17]. PCR reaction (25 μL) was composed of 1x PCR buffer (Mg2+-free), 2 mM MgCl2, 200 μM of each deoxyribonucleoside triphosphate (dNTP), 0.4 μM of each primer, 0.2 U of HotStartTaq DNA polymerase (Takara, Dalian, China), and 2 μL of DNA template. The cycling conditions were 5 min at 95°C for initial denaturation and then 35 cycles of 45 s at 94°C, 45 s at 55°C, and 1 min at 72°C, followed by final extension at 72°C for 10 min. Both positive and negative controls were included in each test. PCR products were observed under UV light after electrophoresis in 1.5% agarose gel containing GoldView™ (Solarbio, Beijing, China).


## 2.4. Sequencing Analyses

The positive PCR products of Java sparrows were sequenced by GenScript Company (Nanjing, China).Cryptosporidium species were determined by alignment with known reference sequences available in GenBank using BLAST (https://www.ncbi.nlm.nih.gov/BLAST/). Representative nucleotide sequences were deposited in GenBank under accession numbers KY034418-KY034427 and KY962159.


## 2.5. Statistical Analysis

The variation inCryptosporidium prevalence (y) of Java sparrows of different geographical location (x1), age (x2), species (x3), and gender (x4) was analyzed by χ2 test using SPSS 19.0. In the multivariable regression analysis, each of these variables was included in the binary logit model as an independent variable. The best model was judged by Fisher’s scoring algorithm. All tests were two-sided, and values of probability (P) < 0.05 were considered statistically significant. Odds ratios (ORs) and their 95% confidence intervals (95% CIs) were estimated to explore the strength of the association betweenCryptosporidium positivity and the conditions tested.


## 3. Results and Discussion

Forty-seven out of 350 Java sparrows (13.42%) were tested to beCryptosporidium-positive by nested PCR amplification of the SSU rRNA gene (Table 1).Cryptosporidium prevalence was 9.82% (16/163) in Beijing and 16.58% (31/187) in Shangqiu (Table 1). The prevalence in white Java sparrows and gray Java sparrows was 16.44% (37/225) and 8.00% (10/125), respectively (Table 1). Moreover, female Java sparrows (40.63%, 26/64) had a statistically higherCryptosporidium prevalence than males (7.34%, 21/286) (Table 1). The infection rates ofCryptosporidium in the different age ranged from 10.47% to 16.33%, with the highest prevalence in the 6–8-month group (Table 1), but the difference was not statistically significant among age groups (P>0.05). Species and sex of Java sparrows were considered as main risk factors to influence the positive rate significantly.Analyses of the obtained SSU rRNA sequences indicated that all of the 47 isolates representedC. baileyi. These sequences were deposited in GenBank under accession numbers KY034418-KY034427 and KY962159. Comparative analyses of all the obtained SSU rRNA sequences with that of the two known reference sequences (C. baileyi, GU816040 andC. baileyi, AF262324) indicated that no variation was found among them.In this study, the prevalence ofCryptosporidium in Java sparrows is 13.42%, which is higher than the 4.86% prevalence in birds in Brazil by PCR [11], 8.1% in white Java sparrows in China by Sheather’s sugar flotation technique [12], and 3.22% in parrots in China by ELISA [2]. Moreover, theCryptosporidium prevalence of the present study was lower than that in pigeons in Thailand (25%) [18]. Different age distributions, test methods, geological environment conditions, and the breeding density may contribute to the difference ofCryptosporidium prevalence [2, 19].In the present study, all of the obtained 47Cryptosporidium isolates belong toC. baileyi.C.baileyi is approximately the most common avianCryptosporidium [20, 21] because it has been reported in a wide range of avian hosts including chickens, turkeys, ducks, black-billed magpie, common myna, crested lark, Gouldian finch, red-billed leiothrix, mixed-bred falcons, zebra finch, rose-ringed parakeet, white Java sparrow, grey partridge, saffron finch, and ruddy shelducks [7, 12, 20–22].


## 4. Conclusion

The present study revealed overallCryptosporidium prevalence of 13.42% (47/350) with 16.44% (37/225) in white Java sparrows and 8.00% (10/125) in gray Java sparrows. This is the first report ofC. baileyi in gray Java sparrows in China, which extend the host forC. baileyi. These results provide baseline information for further studies of molecular epidemiology and control ofCryptosporidium infection in poultry in China.



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*Source: 2318476-2017-07-04.xml*