Low prevalence of Neospora caninum and Toxoplasma gondii antibodies in dogs in Jilin, Henan and Anhui Provinces of the People’s Republic of China

Background Neospora caninum and Toxoplasma gondii are important pathogens of worldwide distribution. N. caninum is a major cause of abortion in cattle and dogs are main reservoirs because they excrete the environmentally resistant oocysts. Toxoplasmosis is a worldwide zoonosis and dogs are considered as sentinels for this parasite because of their close contact with people and cats; additionally dog meat is also used for human consumption in China. The aim of the present study was to assess the prevalence of N. caninum and T. gondii infection in dogs from China. A total of 425 countryside dog hearts in Jilin, Henan and Anhui provinces of the People’s Republic of China were collected from slaughter houses in two batches; the first batch of 96 in October 2013, and the second batch of 329 in April 2014. Serum samples extracted from 96 dog hearts were tested for antibodies to N. caninum and from 425 dog hearts were tested for T. gondii antibodies in the modified agglutination tests (cut-off 1:25 for both), using respective antigens. Results Antibodies to N. caninum were 6 of 96 (6.25%) of dogs with titers of 1:25 in 2, 1:50 in 3, and 1:100 in 1. All seropositive dogs were more than 1 year old. Antibodies to T. gondii were found in 35 of 425 (8.24%) dogs with titers of 1:25 in 15, 1:50 in 14; and 1:100 in 6. Conclusion The results of the present study indicated low prevalence of N. caninum and T. gondii antibodies in dogs of China, compared with Europe and America. Identification of the risk factors that underlie these differences may help prevention of neosporosis and toxoplasmosis. This is the first report of N. caninum infection in dogs from China.


Background
Neospora caninum and Toxoplasma gondii are related coccidians that until 1988 were considered the same organism [1]. N. caninum is now considered the most important abortifacient for cattle worldwide, including China [2,3]. Dog, wolf, coyote and dingo are the definitive hosts for N. caninum that shed environmentally resistant oocysts. Toxoplasma gondii infection in dogs is important for following reasons. Dogs can be infected through contact with the T. gondii, which may be acquired from rooting in infected soil or from ingesting cat feces or from eating raw meat. Dogs can also mechanically transmit T. gondii oocysts to humans [4]. In China, dogs serve as food animals, the consumption of undercooked meat containing T. gondii tissue cysts can be a supplementary health risk to consumers.
Currently, there is no report of isolation T. gondii from dog in China, and little is known of N. caninum infections in dogs in China. The objective of present study was to determine the seroprevalence of N. caninum and T. gondii infections in dogs from China, and to attempt isolate T. gondii.

Naturally infected dogs
A total of 425 countryside dogs were sampled from the slaughter house (Table 1). These countryside dogs in China are part of the farmer's household; they were mainly used for guarding. Their diet includes boiled rice, discarded raw food animal tissues and whatever dogs can forage. These dogs were sold for food. Hearts of dogs were selected for the present study because serum could be obtained from the heart. The first batch samples (96 dog hearts) were collected in October 2013; 50 dogs were younger than 1 year, and 46 were older than 1 year. Second batch samples (329 dog hearts) were collected in April 2014; they were older than 1 year. Blood was collected from heart 1 day (second batch) or three days (first batch) after slaughter, centrifuged at 2000 × g for 10 min, and sera were separated. Hearts samples from first batch were transported by air as part of personal baggage of the senior author from China to the Animal Parasitic Diseases Laboratory (APDL), United States Department of Agriculture, Beltsville, Maryland, USA within one week of killing. During interim, samples were kept cold. Permission papers from USA and China were obtained before transportation.

Ethical aspects
This study was approved by the institutional animal use protocol committee of the United States Department of Agriculture and the Henan Agriculture University, China.

Climatic conditions
The climate of Henan (

Serologic examination
Serum samples from 425 dogs were tested for antibodies to T. gondii using the modified agglutination test (MAT) [5]. Serum samples from first batch samples (96 dogs) were also tested for N. caninum antibodies by the Neospora agglutination test (NAT) [6]. A titer of 1:25 was considered as indicative of exposure to both parasites. Sera were diluted with phosphate-buffered saline and tested 1:25, 1:50, 1:100 and 1:200 dilutions for both parasites. In addition, for T. gondii, sera were also tested at 1:10 dilution.

Bioassay of tissues for T. gondii
Myocardium (50 g, or 30 g for young dog) from the first batch samples with MAT of T. gondii seropositive (MAT, ≥10) dogs (n = 14) was digested individually in pepsin and bioassayed in mice as described [7]. Briefly, tissues were washed and homogenized in saline (0.85% NaCl), mixed with acidic pepsin, and incubated in a shaker water bath for 60 min at 37°C. The homogenate was filtered through two layers of gauze, centrifuged, sediment neutralized with sodium bicarbonate, centrifuged again, mixed with antibiotics, and the homogenate inoculated subcutaneously into three Swiss Webster (SW) outbreed albino mice, and two gamma interferon gene knockout (KO) mice [7]. All inoculated mice were observed daily for illness. Dead mice, or killed when ill, were examined for T. gondii by making impression smears from the lung and examined for tachyzoites. Survivors were bled on day 41 post-inoculation (p.i.) and 1:100 dilution of serum from each mouse was tested for T. gondii antibodies with the MAT. Mice were killed 47 or 48 days post infection and brains of all mice were examined for tissue cysts as a squash preparation as described [7].
Antibodies to N. caninum were found in 6 (13.95%) of 43 dogs from Jilin but not in 53 dogs from the Henan and Anhui, because most of the dogs from other two regions were less than one year old. Antibodies to T. gondii were found in 35 of 425 (8.24%) dogs with titers of 1:25 in 15, 1:50 in 14; and 1:100 in 6, none was positive at 1:200 (Table 1). The age range of the N. caninum and T. gondii negative dogs was 1.06 ± 0.55 (years) and 2.90 ± 1.38 (years). The age range of the N. caninum and T. gondii positive dogs was 2.25 ± 1.42 (years) and 3.06 ± 2.16 (years). T. gondii was not isolated in mice inoculated with tissues of any dogs. Hearts samples and serum were kept cold and transported by air from China to USA within one week of killing. One week antibodies are fairly stable. T. gondii antibodies were stable on dried filter papers for up to 45 days at room temperature [8] and for 6 months at 25°C when stored with silica gel [9]. In an unpublished experiment antibodies were still detectable in blood 7 days after they were accidently left in a water bath at 37°C (JP Dubey, unpublished 1971).
In the present study, antibodies to N. caninum were detected in dog sera for the first time from China. How dogs become infected with N. caninum in nature is unknown [1]. Congenital transmission and ingestion of infected tissues or oocysts are the three probable modes of transmission. Of these, the ingestion of infected tissues is considered the most important because congenital transmission in dogs is relatively rare and fecal transmission has not been proven [3]. Little is known of the natural epidemiology of N. caninum in nature. In the USA, cattle and deer are commonly infected with N. caninum. Viable parasite has not been isolated from small mammals. Little is known of the epidemiology of neosporosis in China. In this study, antibodies to N. caninum were 6.25% (6/96) of first batch samples tested. We didn't test the second batch samples because of non availability of reagents for testing of N. caninum by NAT, or IFA, or cELISA in China. Compared with other countries [10][11][12][13], the prevalence of N. caninum antibody was low in East Asia [14,15]; also it is low in this study. Although a comparison of different serological tests (NAT, IFA, cELISA) for the detection of N. caninum antibodies in dogs has not been made, it is generally accepted that results by NAT and IFA are comparable because both tests assess antibodies directed against whole tachyzoites [1]. None of the serological tests, however, have been validated because it is very difficult to isolate N. caninum from asymptomatic hosts [1].
In this study, the prevalence of antibodies and titers to T. gondii were low compared to data from other parts of China (Table 2, Figure 1) and other country [16,17]. However, results are not strictly comparable because of different serological tests used and different cut-off used. In several of these previous reports, the serological tests used have not been critically evaluated. To facilitate further investigations we have summarized available reports on canine toxoplasmosis in Table 2. The MAT we used has been extensively used for the detection of T. gondii antibodies in many species, including humans and dogs [7]. Viable T. gondii was isolated from 51% (22/43) of dogs with MAT antibodies [18] and there is no evidence for any cross reactivity with other antigens in MAT [19]. The lack of isolation of T. gondii from the hearts of any of the dogs in the present study could be related to the loss of infectivity during transit from China and USA. However, viable T. gondii has been isolated in the USDA laboratory in Beltsville from tissues of animals that had been in transit for up to 10 days from several countries to USA. The survival of T. gondii tissue in meat at room temperature is largely unknown. However, viable T. gondii was isolated from a Hawaiian bird that was completely rotten and most carcasses had been eaten by maggots [20]. At 40°C, T. gondii tissue cysts survived up to 2 months [21]. Temperatures at which the dog tissues samples were stored varied from 4-20°C while in transit from China to USA. Therefore, we are uncertain of the effect of storage on the survival of tissue cysts.

Conclusions
Results of the present study indicated low prevalence of N. caninum and T. gondii infection in dogs in China, compared with data from Europe and America. Identification of the risk factors that underlie these differences may help prevention of neosporosis and toxoplasmosis. Dog meat is consumed by local people in China. Therefore, dogs are at a risk of T. gondii infection and should be of public health concern.
Competing interests None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
Authors' contributions YRY performed the laboratory tests, data analysis, and wrote the manuscript. QFZ participated in the collect samples, and the laboratory tests. YGK participated in the collect samples, obtained the general data of the dogs. YQY participated in the laboratory tests. OCHK participated in the laboratory tests and helped in the writing of the manuscript. HDL designed the study protocol, obtained the serum samples and general data of the dogs. JP D designed the study protocol, analyzed the results and helped in the writing of the manuscript. All authors read and approved the final version of the manuscript.