# Toxoplasma gondii andNeospora caninum in Free-Range Chickens in Henan Province of China

Authors: Yongjie Feng; Yaoyao Lu; Yinghua Wang; Jing Liu; Longxian Zhang; Yurong Yang
Journal: BioMed Research International
 (2016)

---

## Abstract

Background. Chickens serve as an intermediate host forToxoplasma gondii andNeospora caninum; infection of free-range (FR) chickens with these organisms is a useful indicator of soil and environmental contamination with oocysts. A total of 700 FR chicken serum samples and 300 heart samples were collected from Henan province from March to July 2015. Antibodies toT. gondiiwere found in 18.86% (132/700) of the chickens by modified agglutination test (cut-off 1 : 25), while 23.14% (162/700) were positive forN. caninumby indirect fluorescent antibody test (cut-off 1 : 25).T. gondiiDNA was detected in the myocardium digestion liquids of 4/25 (16%) FR chickens. The PCR results ofN. caninum DNA from FR chicken myocardium digestion liquids (n
=
25) were all negative. Attempts to isolate viableT. gondii were unsuccessful. The results showed that there were antibodies toT. gondii andN. caninum in FR chickens from Henan province. Accordingly, effective control of feces from cats and dogs and improved pets hygiene habits were needed. To the author’s knowledge, this is the first report of the presence ofN. caninum antibody in chickens from China.


---

## Body

## 1. Background

Many studies have evaluatedToxoplasma gondiiandNeospora caninuminfections in birds worldwide, including chickens [1, 2]. Chickens contribute to theT. gondii andN. caninum life cycle by acting as intermediate hosts or mechanical vectors [3, 4]. The main route of infection for chickens is assumed to be through ingestion of oocysts from soil. Limited reports of natural neosporosis in birds indicate that they do not show any significant histological lesions [4, 5]. Chickens are resistant to toxoplasmosis, and mostT. gondii positive chickens remain asymptomatic. However, investigation ofT. gondii andN. caninum infection in the free-range (FR) chickens is a useful indicator for assessment of soil and environmental contamination with oocysts.Despite the high body temperature of avians (41°C), they could be infected by toxoplasmosis. Further,T. gondii tachyzoites have been observed in avian red blood cells [1]. Meat from chickens is consumed widely and is known to be the primary source of toxoplasmosis infection for humans. ViableT. gondii have been widely isolated from chicken heart, brain, and muscle in North America and Europe by bioassay in mice or cats [2]. China has the highest number of chickens in the world (approximately 4 billion, https://top5ofanything.com/list/0cffba91/Countries-With-the-Most-Chickens) and an unknown number of wild birds. However, only two viableT. gondii have been isolated from chickens in China [6, 7]. Reports of the genotype and pathogenicity ofT. gondii revealed significant differences among populations in Asia and other parts of the world [2, 8]. Accordingly, further studies to develop methods of isolatingT. gondii from China are warranted.Few English papers have investigated the seroprevalence ofT. gondii in birds from China. Yan et al. summarized the literature (1985–2008) pertaining toT. gondiiprevalence in chickens from China [9]. Although chickens, sparrows, and pigeons have been shown to be intermediate hosts ofN. caninum [4], there is little information regarding theN. caninum seroprevalence in chickens from China. The present study was conducted to investigate the prevalence ofN. caninumandT. gondii infections in FR chickens from central China and to isolateT. gondii.


## 2. Methods

### 2.1. Sera Samples

The study area was Henan province (33.54°N, 113.30°E), which is located in central China. A total of 700 FR chicken sera and 300 fresh hearts from individual farm in Henan province were collected (Figure1 and Table 1). Clinical information (age, gender) regarding all chickens except batch 5 and batch 6 was obtained.Table 1
Prevalence ofT. gondii in free-range chickens from Henan of China.
Batch number
Locationd
City/sample reception date
Number of samples received
Number of seropositive samples (cut-off: 1 : 25, %)
Isolates obtained by mice from heartsa
1
I
Xinyang/9 March 2015
201b
56 (27.9)
—
2
III
Xinxiang/9 March 2015
40
3 (7.5)
—
3
IV
Jiyuan/9 March 2015
68
10 (14.7)
—
4
II
Nanyang/9 March 2015
14
6 (42.9)
—
5
I
Xinyang/8 May 2015
7c
2 (28.6)
0/2
6
I
Xinyang/10 May 2015
51c
12 (23.5)
0/8
7
V
Zhoukou/14 May 2015
62
29 (46.8)
0/15
8
I
Xinyang/20 May 2015
13
0
—
9
I
Xinyang/24 May 2015
14
1 (7.1)
—
10
I
Xinyang/2 June 2015
15
0
—
11
I
Xinyang/9 June 2015
17
0
—
12
I
Xinyang/1 July 2015
7
0
—
13
I
Xinyang/8 July 2015
191b
13 (6.8)
—
Total
700
132 (18.86)
0/25
aNumber of positive groups/number of inoculated groups.
bNo heart tissue available.
cNo detailed information regarding chickens.
dSampling city in Figure 1.Figure 1
Location and number of samples received from Henan province of China. I: Xinyang; II: Nanyang; III: Xinxiang; IV: Jiyuan; V: Zhoukou.


### 2.2. Examination of Chickens Sera forT. gondii andN. caninum Antibodies

Sera samples from 700 FR chickens were tested for antibodies toT. gondii using the modified agglutination test (MAT) [2].T. gondii positive serum andN. caninum positive serum from mice were provided by Dr. J. P. Dubey (ARS, USDA) as reference sera. Whole formalin-treatedT. gondii tachyzoites antigens were obtained from Kerafast Company (catalog number EH2002). Additionally, 700 serum samples were tested forN. caninum antibodies by the indirect fluorescent antibody test (IFAT). The 96-well IFAT plates (whole formalin fixed NC1N. caninum tachyzoites) were kindly provided by Dr. Q. Liu (China Agricultural University, China). The IFAT secondary antibody was goat anti-chicken IgY H&L labeled with Alexa Fluor 488 (Abcam Company, ab150169). Positive control, negative control, and blank were performed on each plate. A titer of 1 : 25 was considered to indicate exposure toT. gondiiin chickens [2, 3]. In addition, all serum samples were tested forT. gondii parasites at 1 : 50, 1 : 100, and 1 : 200 dilutions. IFAT was used to test chicken serumN. caninum antibody (cut-off: 1 : 25), and only bright fluorescence of the whole tachyzoite surface was considered as a positive result [10].


### 2.3. Isolation of ViableT. gondii from Chicken Tissues by Bioassay ofKunming Mice

Chicken myocardia (10 g) from the samples with MAT ofT. gondii seropositive (MAT, ≥50) chickens (n
=
25) were digested individually in pepsin and bioassayed inKunming(KM) outbred mice as previously described [2]. Each heart homogenate was inoculated subcutaneously into two mice. All inoculated mice were observed daily for illness. Dead mice were examined forT. gondii by making impression smears from the lung or brain, which were inoculated into a new group of mice regardless of whether they were negative or positive. Survivors were bled on 42 days postinoculation (DPI) and 1 : 25, 1 : 200 dilution of serum from the mouse was tested forT. gondii antibodies using the MAT. Mice were killed after 49 DPI, and brains of all mice were examined for tissue cysts and inoculated into new groups of mice. The sheep hearts containingT. gondiicysts were used as protocol control.


### 2.4. DNA Isolation and Polymerase Chain Reaction (PCR) Identification ofT. gondii andN. caninum

Chickens myocardium digestion liquids from chicken hearts were also used to detectT. gondii DNA. The DNA isolated fromT. gondii (CT1 strain) orN. caninum (NC1 strain) was used as a reference for PCR. CT1 strainT. gondii was kindly provided by Dr. J. P. Dubey (ARS, USDA). NC1 strainN. caninumwas kindly provided by Dr. Q. Liu (China Agricultural University, China). The DNA of digestion heart samples was extracted using a commercial DNA extraction kit (Tiangen Biotec Company, DP304). PCR assays forT. gondii andN. caninumwere performed using the specific primer pairs TOX5/TOX8 (5′-CGCTGCAGACACAGTGCATCTGGATT-3′ and 5′-CCCAGCTGCGTCTGTCGGGAT-3′) and NP6/NP21 (5′-CAGTCAACCTACGTCTTCT-3′ and 5′-GTGCGTCCAATCCTGTAAC-3′) [11, 12]. The products fromT. gondiiwere expected to be 450 bp.N. caninumexpected products were 328 bp. Briefly,T. gondii reaction cycle was as follows: initial denaturation at 94°C for 2 min, followed by 35 cycles of amplification (94°C for 1 min, 60°C for 1 min, and 72°C for 1 min) and then final extension at 72°C for 10 min.N. caninumreaction cycle was as follows: initial denaturation at 94°C for 3 min, followed by 35 cycles of amplification (94°C for 1 min, 53°C for 1 min, and 72°C for 2 min) and then final extension at 72°C for 10 min.


### 2.5. Statistical Analysis

Statistical analysis was performed by GraphPad Prism 4.0 software (GraphPad Software Inc., San Diego, CA, USA). Data were analyzed by the Chi-square test or Fisher’s exact test to determine the association between infection with each parasite and independent risk factors: gender (male and female) and age (≤30 days, >30 days).p
<
0
.
05 was considered statistically significant.


## 2.1. Sera Samples

The study area was Henan province (33.54°N, 113.30°E), which is located in central China. A total of 700 FR chicken sera and 300 fresh hearts from individual farm in Henan province were collected (Figure1 and Table 1). Clinical information (age, gender) regarding all chickens except batch 5 and batch 6 was obtained.Table 1
Prevalence ofT. gondii in free-range chickens from Henan of China.
Batch number
Locationd
City/sample reception date
Number of samples received
Number of seropositive samples (cut-off: 1 : 25, %)
Isolates obtained by mice from heartsa
1
I
Xinyang/9 March 2015
201b
56 (27.9)
—
2
III
Xinxiang/9 March 2015
40
3 (7.5)
—
3
IV
Jiyuan/9 March 2015
68
10 (14.7)
—
4
II
Nanyang/9 March 2015
14
6 (42.9)
—
5
I
Xinyang/8 May 2015
7c
2 (28.6)
0/2
6
I
Xinyang/10 May 2015
51c
12 (23.5)
0/8
7
V
Zhoukou/14 May 2015
62
29 (46.8)
0/15
8
I
Xinyang/20 May 2015
13
0
—
9
I
Xinyang/24 May 2015
14
1 (7.1)
—
10
I
Xinyang/2 June 2015
15
0
—
11
I
Xinyang/9 June 2015
17
0
—
12
I
Xinyang/1 July 2015
7
0
—
13
I
Xinyang/8 July 2015
191b
13 (6.8)
—
Total
700
132 (18.86)
0/25
aNumber of positive groups/number of inoculated groups.
bNo heart tissue available.
cNo detailed information regarding chickens.
dSampling city in Figure 1.Figure 1
Location and number of samples received from Henan province of China. I: Xinyang; II: Nanyang; III: Xinxiang; IV: Jiyuan; V: Zhoukou.


## 2.2. Examination of Chickens Sera forT. gondii andN. caninum Antibodies

Sera samples from 700 FR chickens were tested for antibodies toT. gondii using the modified agglutination test (MAT) [2].T. gondii positive serum andN. caninum positive serum from mice were provided by Dr. J. P. Dubey (ARS, USDA) as reference sera. Whole formalin-treatedT. gondii tachyzoites antigens were obtained from Kerafast Company (catalog number EH2002). Additionally, 700 serum samples were tested forN. caninum antibodies by the indirect fluorescent antibody test (IFAT). The 96-well IFAT plates (whole formalin fixed NC1N. caninum tachyzoites) were kindly provided by Dr. Q. Liu (China Agricultural University, China). The IFAT secondary antibody was goat anti-chicken IgY H&L labeled with Alexa Fluor 488 (Abcam Company, ab150169). Positive control, negative control, and blank were performed on each plate. A titer of 1 : 25 was considered to indicate exposure toT. gondiiin chickens [2, 3]. In addition, all serum samples were tested forT. gondii parasites at 1 : 50, 1 : 100, and 1 : 200 dilutions. IFAT was used to test chicken serumN. caninum antibody (cut-off: 1 : 25), and only bright fluorescence of the whole tachyzoite surface was considered as a positive result [10].


## 2.3. Isolation of ViableT. gondii from Chicken Tissues by Bioassay ofKunming Mice

Chicken myocardia (10 g) from the samples with MAT ofT. gondii seropositive (MAT, ≥50) chickens (n
=
25) were digested individually in pepsin and bioassayed inKunming(KM) outbred mice as previously described [2]. Each heart homogenate was inoculated subcutaneously into two mice. All inoculated mice were observed daily for illness. Dead mice were examined forT. gondii by making impression smears from the lung or brain, which were inoculated into a new group of mice regardless of whether they were negative or positive. Survivors were bled on 42 days postinoculation (DPI) and 1 : 25, 1 : 200 dilution of serum from the mouse was tested forT. gondii antibodies using the MAT. Mice were killed after 49 DPI, and brains of all mice were examined for tissue cysts and inoculated into new groups of mice. The sheep hearts containingT. gondiicysts were used as protocol control.


## 2.4. DNA Isolation and Polymerase Chain Reaction (PCR) Identification ofT. gondii andN. caninum

Chickens myocardium digestion liquids from chicken hearts were also used to detectT. gondii DNA. The DNA isolated fromT. gondii (CT1 strain) orN. caninum (NC1 strain) was used as a reference for PCR. CT1 strainT. gondii was kindly provided by Dr. J. P. Dubey (ARS, USDA). NC1 strainN. caninumwas kindly provided by Dr. Q. Liu (China Agricultural University, China). The DNA of digestion heart samples was extracted using a commercial DNA extraction kit (Tiangen Biotec Company, DP304). PCR assays forT. gondii andN. caninumwere performed using the specific primer pairs TOX5/TOX8 (5′-CGCTGCAGACACAGTGCATCTGGATT-3′ and 5′-CCCAGCTGCGTCTGTCGGGAT-3′) and NP6/NP21 (5′-CAGTCAACCTACGTCTTCT-3′ and 5′-GTGCGTCCAATCCTGTAAC-3′) [11, 12]. The products fromT. gondiiwere expected to be 450 bp.N. caninumexpected products were 328 bp. Briefly,T. gondii reaction cycle was as follows: initial denaturation at 94°C for 2 min, followed by 35 cycles of amplification (94°C for 1 min, 60°C for 1 min, and 72°C for 1 min) and then final extension at 72°C for 10 min.N. caninumreaction cycle was as follows: initial denaturation at 94°C for 3 min, followed by 35 cycles of amplification (94°C for 1 min, 53°C for 1 min, and 72°C for 2 min) and then final extension at 72°C for 10 min.


## 2.5. Statistical Analysis

Statistical analysis was performed by GraphPad Prism 4.0 software (GraphPad Software Inc., San Diego, CA, USA). Data were analyzed by the Chi-square test or Fisher’s exact test to determine the association between infection with each parasite and independent risk factors: gender (male and female) and age (≤30 days, >30 days).p
<
0
.
05 was considered statistically significant.


## 3. Results and Discussion

Antibodies toT. gondiiwere found in 18.86% (132/700) of FR chickens from Henan province of China (Table 1). Seropositivity rates varied with respect to gender, with antibodies being found in 33.59% of males and 14.48% of females (p
<
0
.
01) (Tables 2 and 3). This was probably due to a greater probability of exposure toT. gondii oocysts in males because of the active characteristic of roosters. Many studies indicated that gender was not a risk factor for toxoplasmosis [1, 13]. However,T. gondii change the concentration of steroid hormones in the host and enhance the susceptibility of males or females to toxoplasmosis; it was decided by the stainsT. gondii [14, 15]. A high percentage of male sheep were also positive forT. gondii antibody based on serological survey of China (p
>
0
.
05) (Feng et al., unpublished data).Table 2
Seroprevalence ofT. gondiiand N. caninum infection in free-range chickens.
Characteristics
Number tested
Positive number in different titers
Seroprevalence %  (positive number)
95% CI
1 : 25
1 : 50
1 : 100
Above 1 : 200
T. gondii (MAT, cut-off: 1 : 25)
Gender: female
511
46
10
4
14
14.48  (74)
11.68–17.81
Gender: male
131
37
2
2
3
33.59  (44)
26.06–42.05
Age (days) ≤ 30
82
10
0
0
3
15.85  (13)
9.37–25.40
Age (days) > 30
560
73
12
6
14
18.75  (105)
15.73–22.20
No informationa
58
9
0
0
5
24.14 (14)
14.85–36.64
Total
700
92
12
6
22
18.86  (132)
16.13–21.93
N. caninum (IFAT, cut-off: 1 : 25)
Gender: female
511
123
—
—
—
24.07  (123)
20.56–27.97
Gender: male
131
27
—
—
—
20.61  (27)
14.52–28.38
Age  (days) ≤ 30
82
21
—
—
—
25.61  (21)
17.34–36.06
Age  (days) > 30
560
129
—
—
—
23.04  (129)
19.73–26.70
No informationa
58
12
—
—
—
20.69 (12)
12.10–32.92
Total
700
162
—
—
—
23.14 (162)
20.17–26.41
aNo detailed information  (gender, age) regarding chickens.Table 3
Odds ratio of gender and age of chickens as risk factors forT. gondiiand N. caninum.
Factor
Category
OR
95% CI
p value
T. gondii
Gender
Female
—
—
—
Male
2.987
1.926–4.630
p
<
0.0001
∗
Age (days)
≤30
—
—
—
>30
1.225
0.653–2.299
p
=
0.6471
N. caninum
Gender
Female
—
—
—
Male
1.221
0.764–1.953
p
=
0.4874
Age (days)
≤30
—
—
—
>30
1.150
0.675–1.961
p
=
0.5795
OR: odds ratio; “∗” indicates significant difference.The seroprevalence ofT. gondii is age related, with higher seroprevalence being observed in older chickens (>30 days, 18.75%) than in younger chickens (≤30 days, 15.85%); however, this difference was not significant (Tables 2 and 3). It is worth noting that 15.85% of 82 young chickens were positive forT. gondiiantibody, indicating maybe vertical transmission from embryos.Among the 700 FR chickens tested forN. caninum antibodies, 23.14% (162/700) were positive (Tables 2 and 3). The PCR results ofN. caninum DNA from FR chicken myocardium digestion liquids (n
=
25) were all negative. Age and gender showed no association with seroprevalence (p
>
0
.
05). Additionally, 3.57% (25/700) FR chickens mix was exposed to theT. gondii andN. caninum. Moreover, 25.61% (21/82) of younger chickens (<30 days) were seropositive forN. caninum. An epidemiological survey of cattle and dogs supported the postnatal transmission ofN. caninum[16, 17]. The high seropositive rate in younger chickens indicated thatN. caninum transmission possibly occurs via eggs in birds. However, vertical transmission byN. caninum in chickens was not detected by microscopy, PCR, or BALB/c mice bioassays [18]. The methods of microscopy and PCR were specific and sensitive, but there were limitations for only checking few samples. It was temporal tissue distribution and parasite loads duringN. caninum infection in BALB/c murine model [19]. Gerbils, immunodeficient mice, and cell cultures were the successful model for isolatingN. caninum from tissues [20]. The exact evidence forN. caninum infection from hen to egg needs to be further investigated and proved.T. gondii was not isolated from the mice inoculated with tissues of any chickens hearts in this study. The sheep hearts containingT. gondiicysts were used as protocol control. This excludes the errors of the protocol, such as pH of the solutions, enzymatic activity, and period of digestion. It has been shown that the density ofT. gondii cysts in hearts is higher than in brain or muscle in chicken, and the heart is the ideal choice for isolation ofT. gondii [2, 21].T. gondii bioassays in cats are more sensitive than in mice. The use of gamma interferon gene knockout mice or immunosuppressed mice facilitates early detection ofT. gondii because most strains ofT. gondii are asymptomatic in outbred mice. Sreekumar did not isolate any viableT. gondii from 186 (133 MAT seropositive) chicken tissues from India by mouse bioassay [22]. However, Qian successfully isolatedT. gondii (11 isolations from 23 cats tissues) using immunosuppressedKunming mice [23].T. gondii could grow efficiently in macrophages of rats treated with glucocorticoide [24]. The unsuccessful isolation may be related to the low density ofT. gondiicysts burden in chickens of different regions or the natural resistance toT. gondii infection in some strains of mice. PCR indicated that the presence ofT. gondii DNA was 16% (4/25) in FR chicken myocardium digestion liquids, which yielded PCR product of approximately 450 bp. The low infection rate observed upon molecular analysis verified the light burden ofT. gondii cysts in chickens from central China.


## 4. Conclusions

Our results showed that there were antibodies toT. gondii andN. caninum in FR chickens from Henan province of China. The contamination environment was the source of infection with toxoplasmosis for humans and other animals. Accordingly, effective control of feces from cats and dogs and improved pets hygiene habits were needed.



---

*Source: 8290536-2016-05-05.xml*