Toxoplasma gondii and Neospora caninum in farm-reared ostriches (Struthio camelus) in China

Background The parasites Toxoplasma gondii (T. gondii) and Neospora caninum (N. caninum) are globally distributed; they infect warm-blooded animals, including many avian species. The aim of this study was to evaluate the presence of these parasites in ostriches from central China. In total, 402 ostrich (Struthio camelus) samples (293 hearts, 77 brains, and 32 serum) from slaughterhouses of the Henan Province and Hebei Province were collected. The heart juice (n = 283) and serum samples (n = 32) were tested for antibodies to T. gondii using the modified agglutination test (MAT). Hematoxylin and eosin (H&E) staining, immunohistochemical (IHC) staining, and the polymerase chain reaction were used to examine the cysts and DNA of T. gondii and N. caninum parasites, respectively. Results Antibodies to T. gondii were detected in 6.4% (20/315) (cut-off, 25). No cysts or DNA of T. gondii or N. caninum were observed in any of the 293 hearts and 77 brains. Conclusion The results showed a low prevalence of T. gondii antibody in ostriches, compared to that in the other animals. N. caninum occurs at low to negligible frequencies in ostriches from China. This is the first report on screening ostriches in China for T. gondii antibodies.


Background
Toxoplasma gondii is an obligate cyst-forming parasite known to infect many species of warm-blooded animals, including ostrich [1,2]. Infection with T. gondii is usually asymptomatic, but it can cause severe illness in both humans and animals with weakened immune systems, and in the case of pregnancy [3]. Neospora caninum is a parasite similar to T. gondii in many respects as it causes abortion in cattle and paralysis in dogs [4,5].
The breeding of ostriches for eggs, feathers, skin and meat is growing in China and its meat, in particular, is low in fat and highly palatable. The prevalence of T. gondii in free-range chickens is a good indicator of the presence of oocysts in the environment [6], and ostrich is much like free-range chickens that feed directly from the ground. It was speculated that T. gondii and N. caninum infection in ostriches may assess the presence of oocysts in the local soil and environment. Furthermore, the consumption of undercooked ostrich meat containing T. gondii cysts might be a source of infection to consumers. Although the prevalence of T. gondii in ostriches has been reported in various countries [7][8][9][10][11][12][13], no research studies on T. gondii infection in ostriches from China have been conducted to date. Recently, the first report of isolation of T. gondii from ostriches was published [2]. The aim of this study was to determine the presence of T. gondii and N. caninum in farm-raised ostriches from China and attempt to isolate T. gondii.

Sample collection
A total of 402 ostrich (Struthio camelus) samples (283 fresh hearts, 77 fresh brains, 32 fresh sera, and 10 formalin-fixed hearts) from slaughterhouses in two provinces (Henan Province: 165 hearts, 77 brains, 32 sera and 10 formalin-fixed hearts samples; Hebei Province: 118 hearts) were collected between 2012 and 2015 (Table 1). All ostriches was above 10 months old, however the sex was unknown. The provinces of Henan (31.38°to 36.37°N and 110.35°to 116.65°E) and Hebei (36.08°to 42.67°N and 113.45°to 119.83°E) are located in central China. Unfrozen and fresh samples were transported to the Laboratory of Veterinary Pathology, Henan Agricultural University in cooler boxes. The ostriches were bred in paddocks and fed from the ground with greens and nutritious additives. Heart juice from 283 hearts was collected and centrifuged (1000 g, 5 min) and then assayed for antibodies against T. gondii. A total of 315 samples (283 heart juice and 32 sera) were screened for T. gondii antibodies. The samples were kept in a refrigerator with a temperature range of 2°C to 4°C until analysis (within 1 week).

Screening of ostriches body fluids for T. gondii antibodies
Using the modified agglutination test [14] at a dilution of 1:25, 1:50, 1:100 and 1:200, 315 body fluid samples (283 heart juice and 32 sera) were screened for T. gondii antibodies. T. gondii-positive serum from mice was used as a reference; negative serum and blank were performed in each plate. Whole formalin-treated T. gondii tachyzoite antigen was obtained from the Kerafast Company (Boston, MA, USA. Catalog No. EH2002). Serum dilution of 1:25 is effective for assessing the T. gondii antibody, which has been reported in ostriches and other animals [7,8].
Histopathological examination of ostrich heart and brain tissues for T. gondii and N. caninum A total of 370 ostrich tissue samples (293 hearts and 77 brains) were respectively cut into three pieces (1.0 × 1.0 × 0.3 cm), embedded in paraffin, and sectioned at a thickness of 5 μm. These tissue sections were then stained with hematoxylin and eosin (H&E). Immunohistochemical (IHC) staining was used to verify suspected samples by rabbit polyclonal T. gondii antibody and mouse polyclonal N. caninum antibody. T. gondii-positive tissues or N. caninum-positive tissues from mice were used as reference; negative serum and blank were performed in each batch. The sections were examined with an Olympus CX21 optical microscope to search for cysts and determine the infection of T. gondii or N. caninum.

Isolation of viable T. gondii from ostrich hearts by bioassay in mice
Ostrich hearts that tested positive for T. gondii antibodies were bioassayed in mice individually (n = 8); sheep hearts containing T. gondii cysts were used as controls. Myocardia (50 g) from each heart were homogenized, digested in pepsin, and inoculated subcutaneously into 5 outbred Kunming mice, which were given water supplemented with dexamethasone phosphate (10 μg/ml) 3 days before inoculation [3]. The remaining pepsin-digested myocardial samples were stored in 1.5 mL cryotubes at −20°C until further analysis. Clinical signs in these mice were observed daily. The mice were bled and sacrificed at 64 days post inoculation. Then the sera were diluted 1:25 and 1:200 to test for T. gondii antibodies; in addition, squash preparations of the brain were microscopically examined for T. gondii cysts. The mouse brains were then inoculated into new groups of mice.
PCR identification of T. gondii and N. caninum in ostrich heart and brain tissues DNA was extracted from all tissue samples (293 hearts, 77 brains, and 8 pepsin -digested sediments of the myocardium) using a commercial DNA extraction kit (Tiangen Biotec Company, Beijing, China, DP304). PCR was used to detect the DNA of T. gondii (TOX5/TOX8) in amplified fragments of 450 bp [15] and N. caninum (NP6/NP21) in fragments of 337 bp [16]. The DNA isolated from T. gondii (CT1 strain) or N. caninum (NC1 strain) was used as a reference for PCR.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA,  [24]. Compared with these reports, seroprevalence of T. gondii was lower among farm-reared ostriches (10.2%, 20/197) in the same location. This result is consistent with other reports. A low prevalence of T. gondii antibodies in ostriches has been reported in other countries. The seroprevalence of T. gondii was about 74% to 80% among backyard chickens in Brazil [25,26], but lower among ostriches (about 11% to 17%) [2,9,10] (Table 2). A summary of the prevalence of T. gondii in ostriches in various countries is shown in Table  2. Prevalence varies from 1% to 48%, depending on the country. In conclusion, this is the first report to show the prevalence of T. gondii in ostriches from China.
T. gondii isolation, T. gondii and N. caninum morphological and molecular assays Viable T. gondii had been isolated from ostrich brains, thereby serving as direct evidence that ostriches are intermediate hosts [2]. Isolation of T. gondii from the chicken heart or brain by bioassay in mice has been shown to be effective [3,27]. A previous study has shown that the density of T. gondii cysts in the heart is higher than that in muscle or brain of chickens [27]. Eight ostrich hearts were bioassayed in immunosuppressed Kunming mice in an effort to isolate T. gondii, but this was unsuccessful. Furthermore, H&E or IHC staining did not detect any tissue cysts of T. gondii or N. caninum, and no positive DNA of T. gondii or N. caninum was detected by PCR in a total of 370 ostrich tissue samples and 8 pepsin-digested liquids from myocardia. These results may be explained by (1) the relative low density of T. gondii cysts in ostriches from China or (2) false positive on the MAT. Antigen from whole formalin-treated tachyzoites was used in the MAT. The MAT is highly sensitive and has been extensively used to test T. gondii antibodies in many animals and birds, including ostriches. The accuracy of the MAT has been validated in ostriches because 14/38 (36.8%) isolation has been achieved [2]. However, we can not rule out the possibility that T. gondii antigen cross-reacts with other parasites (Hammondia hammondi) [28]. Some birds (chickens, pigeons, sparrows) have been shown to be intermediate hosts for N. caninum [29]. However, a recent literature search has found no study on N. caninum in ostriches. Ostriches have not been proven to be intermediate hosts for N. caninum. An examination of tissue sections was the most straightforward way of observing the parasites and lesions by light microscopy. Mineo found that no serological positivity of N. caninum was observed in 294 samples of serum. However, N. caninum cysts were found in Psittaciformes muscle by tissue section [30]. PCR is a sensitive method for detecting parasites. The DNA of N. caninum in the heart and brain of many animals has detected by PCR, particularly in the brains [31]. The absence of N. caninum from these ostrich samples suggests that this parasite occurs at low to negligible frequencies in ostriches from China. Further investigations using samples from different areas may facilitate in better understanding the process of neosporosis in ostriches.

Conclusions
The results of the present study indicate that T. gondii has a lower prevalence in ostriches compared to other animals, the cause of this difference is unknown. However, consumers are precautions of eating raw or undercooked ostrich meat to avoid being infected with T. gondii. N. caninum occurs at low to negligible frequencies in ostriches from China. The establishment of differences in prevalence rates between ostrich and other birds may assist in the design of preventive and control measures against toxoplasmosis.

Availability of data and materials
The data, materials, and protocols associated with this report may be shared by the readers without undue qualifications.
Authors' contributions YJF performed the laboratory tests, data analysis, and wrote the manuscript. YYL, YHW participated in sample collection and laboratory testing. LXZ helped in the writing of the manuscript. YRY designed the study protocol, analyzed the results and helped in the writing of the manuscript. All authors have read and approved the final version of the manuscript.

Ethics approval
Ethical treatment of animals used in this study was approved by the Institutional Animal Use Committee of Henan Agricultural University (China). The protocol was approved by the Beijing Association for Science and Technology (SYXK [Beijing] 2007-0023).