Seroprevalence of Toxoplasma gondii and Encephalitozoon cuniculi among domestic rabbits in central China

Rabbits (Oryctolagus cuniculus) are frequently reared for meat production in China. The aim of this study was to assess the seroprevalence of Toxoplasma gondii and Encephalitozoon cuniculi, and risk factors of infection in domestic rabbits raised in Henan province, central China. 1,213 serum samples of domestic rabbits were collected and tested for anti-T. gondii and anti-E. cuniculi antibodies using a modified agglutination test (MAT) and an enzyme-linked immunosorbent assay (ELISA), respectively. The serum positive rates of T. gondii and E. cuniculi were 128/1,213 (10.55%) and 235/1,213 (19.37%), respectively. Co-infection of T. gondii and E. cuniculi was demonstrated in 84 specimens; 44 rabbits were seropositive for T. gondii alone, while 151 rabbits were seropositive for E. cuniculi alone. The main risk factors simultaneously associated with T. gondii and E. cuniculi infection were the age of the rabbit, the type of food, and the rabbit rearing system. Serum positive rates of T. gondii and E. cuniculi among domestic rabbits were high, indicating the possibility of public health issues.


Introduction
Toxoplasma gondii (T. gondii) is one of the most common universal zoonotic protozoan parasites, ubiquitous throughout the world, and widely prevalent in humans and animals [8]. It is estimated that up to one-third of the world's human population has been infected with T. gondii [10]. Almost all warm blooded animals are infected by T. gondii, including rabbits [7]. The main route for humans to acquire T. gondii infection is the consumption of raw or undercooked meat containing tissue cysts from intermediate hosts [8].
The transmission mode of T. gondii to rabbits is through water or food containing oocysts from feline excrement, or through placenta from pregnant females to offspring [8]. Humans may become infected not only by eating undercooked rabbit meat but also from contaminated hand-to-mouth processes after slaughtering, skinning rabbits or dealing with undercooked or raw rabbit meat [4,9].
Encephalitozoon cuniculi (E. cuniculi) is a common pathogen of rabbits causing chronic renal and central nervous system disease characterized by granuloma formation and fibrosis [16]. However, it also affects other mammals, including rodents, herbivores, carnivores, nonhuman primates, and humans [1,6]. Infected rabbits eliminate the spores in urine and feces. Humans become infected principally by intake of water or food contaminated with infective spores. Human encephalitozoonosis is mostly found in immunocompromised patients, including organ transplant recipients, HIV patients, and cancer patients who are being treated with chemotherapy [2]. In many rabbits, the infection can persist subclinically for a long period of time [16].
In China, rabbits are frequently reared for meat production and are consequently considered a possible source of infection by T. gondii and E. cuniculi in humans. However, little is known about natural infection of T. gondii and E. cuniculi in domestic rabbits in China. Therefore, this study was conducted to investigate the seroprevalence and the risk factors related to seropositivity of T. gondii and E. cuniculi among domestic rabbits in Henan province, central China. The results will lay the groundwork for controlling both T. gondii and E. cuniculi infections among domestic rabbits in central China.

Ethical statements
All protocols in this study were reviewed and approved by the Ethics Committee of the Xinxiang Medical University (reference no. 2015018).

Collecting samples
A total of 1213 blood specimens of domestic rabbits were collected from the seven above-mentioned cities within Henan province during the period from June 2015 to December 2016. Information on location, species, gender, ages of rabbits as well as the feeding conditions of respective rabbits was recorded. Serum specimens prepared by centrifuging whole blood were subsequently transferred into 1.5 ml Eppendorf tubes and preserved under the temperature of À80°C before being tested against anti-E. cuniculi and T. gondii antibodies.

Determining anti-T. gondii antibody
Based on previous studies, the anti-T. gondii antibodies in serum specimens were determined using the modified agglutination test (MAT) [8,13,21]. T. gondii whole cell antigen (formalin-fixed whole tachyzoites) prepared using the RH strain of T. gondii cultivated via human foreskin fibroblast cells was purchased from KeraFAST, Inc. (Boston, MA, USA). Briefly, serum samples were diluted by serum dilution buffer using 2-fold serial dilutions from 1:25 to 1:3,200. Fifty microlitres of diluted serum samples were used for agglutinating with 50 ml of antigen mixture (mixture of antigen dilution buffer, 2-mercaptoethanol, Evans blue dye solution, and T. gondii whole cell antigen) in a U-bottom 96-well microtiter plate under the temperature of 37°C for a whole night. The formation of parasite agglutinating layers in wells which contained diluted serum specimens with a ratio of 1:25 or higher indicated positive results. Each assay contained negative and positive control groups.

Determination of antibodies against E. cuniculi
The anti-E. cuniculi antibody levels of serum samples from domestic rabbits were verified and detected using a commercial Encephalitozoon cuniculi (EC) ELISA kit (Medicago, Uppsala, Sweden), following the manufacturer's instructions.

Statistical analysis
Chi square tests were used to analyse the variation of serum positive rates for T. gondii and E. cuniculi resulting from variates including species, gender, ages, the type of food, and the rearing system. Windows version SPSS 20 (SPSS Inc, Chicago, IL, USA) was used to perform all statistical analyses. A p-value lower than 0.05 was regarded as the threshold for statistically significant differences.

Seroprevalence of T. gondii
As shown in Table 1, the overall recorded serum positive rate for T. gondii among rabbits from Henan province, central China was 10.55% (128/1213), with titers of 1:25 in 69, 1:50 in 36, 1:100 in 14, 1:200 in 5, 1:400 in 3, and 1:800 in 1. The serum positive rates of 7 sampling sites were within the range of 7.64% in Sanmenxia city to 14.06% in Luoyang city (Table 1). New Zealand rabbits exhibited the maximum value of 12.34%, followed by Chinese rabbits (10.73%) and then Japanese White rabbits (10.58%), while the prevalence found in Rex rabbits was 9.17%.
The rate of antibodies to T. gondii in male rabbits was 11.42% (61/534) and in female rabbits 9.87% (67/679) ( Table 1). Although the seroprevalence in males was higher than the females, the difference was not significant (p>0.05). The prevalence of T. gondii infection in rabbits increased significantly (p < 0.01) with age. Rabbits no younger than twelve months exhibited the highest seroprevalence (17.07%), followed by rabbits of 6-12 months (10.40%), and then rabbits 6 months (6.65%) ( Table 1).
The highest seroprevalence of T. gondii infection (15.99%) was found in rabbits fed with a mixture of fruits, vegetables or grains. The probability of rabbits being infected by T. gondii was increased by the presence of cats at the feeding farm compared to farms without cats (12.52% vs 7.45%, p < 0.01). In terms of rearing systems, the T. gondii seroprevalence in rabbits raised on commercial farms (6.01%) was significantly lower than that of animals raised on household farms (14.31%, p < 0.01).
The univariate analyses on the correlation between serum positive rates of T. gondii and possible risk factors are shown in Table 1, indicating significant associations between serum positive rates of T. gondii and food type, rabbit ages, cat presence on feeding farms, as well as the rearing system.

Seroprevalence of E. cuniculi
As shown in Table 2, in total 235 (19.37%) rabbits were positive for anti-E. cuniculi antibodies. Rabbits with positive antibodies in serum were found at all tested sites, varying from 11.83% in Xuchang city to 29.55% in Zhoukou city (Table 2). Rex rabbits exhibited the highest value of serum positive rates in all studied species (24.16%), followed by New Zealand rabbits (17.41%), then Japanese White rabbits (16.93%), while the prevalence   found in Chinese rabbits was 15.32%. The serum positive rate of female rabbits (21.50%) was remarkably higher than that of male rabbits (16.67%) (p < 0.05). Moreover, it was revealed by comparing infection rates by ages that 39/316 E. cuniculi seropositive animals (12.34%) were young ( 6 months), 135/692 (19.51%) were adults (6∼12 months), and 61/205 (29.76%) were in older age groups (≥ 12 months). The E. cuniculi seropositivity of rabbits increased significantly (p < 0.01) with the increase of rabbit's age.
Like in T. gondii infection, the highest E. cuniculi seroprevalence (27.89%) was found in rabbits fed with a mixture of fruits, vegetables or grains. Moreover, E. cuniculi seroprevalence in rabbits raised on commercial farms was also significantly lower than that of animals raised on household farms (12.93% vs 24.70%, p < 0.01).

Discussion
The current investigation showed that the total serum positive rate of T. gondii was 10.55% among domestic rabbits in Henan, central China, which was lower than that found in Shanghai (23.4%) [22], but higher than that observed in Liaoning (6.5%), Jilin (4.5%), Heilongjiang (3.7%), and the Inner Mongolia Autonomous Region (3.5%) [13] of China. Meanwhile, the serological results of this study revealed that the overall serum positive rate for E. cuniculi was 19.37% in domestic rabbits in Henan. In comparison to other provinces within China mainland, the serum positive rate of 19.37% was lower than the values of 22.2% among rabbits investigated previously in Inner Mongolia, 30.9% in Liaoning [13], and 41.0% in Jilin [15], but higher than that observed in Sichuan (9.0%), Chongqing (6.0%) [15], and Heilongjiang (17.3%) [13]. The variations of serum positive rates for anti-T. gondii and anti-E. cuniculi antibodies among different regions are probably attributable to different rabbit breeds, sample capacities, time of investigations, and testing methods, as well as geographical and ecological factors.
The serum positive rate for T. gondii in the current work was lower than that of E. cuniculi, which was consistent with previous literature reports [13,14]. The spreading of E. cuniculi among herds was remarkably promoted by directly transmitting spores from urine of affected animals [14].
In the present study, the highest seroprevalence of T. gondii was found in New Zealand rabbits, while Rex rabbits had the highest seroprevalence of E. cuniculi. Regarding the breed of rabbits, de Lima et al. [5] and Alvarado-Esquivel et al. [3] reported a higher seroprevalence of T. gondii in New Zealand rabbits than in other breeds. Pan et al. also revealed higher serum positive rates for E. cuniculi among Rex rabbits than those among New Zealand and Japanese White Rabbits (p < 0.01) [15]. These results indicate that there may be a potential association between the breed of rabbits and the seropositivity against T. gondii and E. cuniculi. Most epidemiological surveys indicated no association between T. gondii and E. cuniculi infection and gender of the animals [13,18,19]. In our study, the gender of rabbits was not a significant risk factor for the presence of infection with T. gondii, which was in agreement with many previous studies [5,18]. However, female rabbits exhibited remarkably higher seroprevalence for E. cuniculi in comparison to male animals of our study. The role of gender in the epidemiology of rabbit encephalitozoonosis requires further research.
Increasing age was confirmed as a risk factor for both T. gondii and E. cuniculi infection. The observed higher seroprevalence for both protists in older rabbits suggests that these infections are mainly maintained by horizontal rather than vertical transmission. This finding is in accordance with other similar surveys [13,17].
Rabbits fed with fruits, vegetables and grains had the highest serum positive rates of T. gondii and E. cuniculi according to the current study, which was consistent with other reports [3]. This might be because these foods had a higher level of contamination of T. gondii oocysts or E. cuniculi spores than feed or mixed food. Cats play an essential role in T. gondii spreading since they are the final hosts and shed oocysts into the environment [11,20]. In this study, the presence of cats on rabbit farms was confirmed as one of the risk factors for occurrence of T. gondii infection among rabbits, which accorded with findings in previous literature reports [5,9].
In terms of rearing systems, both T. gondii and E. cuniculi seroprevalence rates were significantly higher in rabbits raised on household farms than in those raised on commercial farms, which is probably due to lower hygiene standards on household farms. Our findings are similar to those of previous reports [12,14]. These results suggest that the rearing system is a very important risk factor associated with T. gondii infection in rabbits as well as E. cuniculi infection.