Molecular and serological prevalence of Toxoplasma gondii and Anaplasma spp. infection in goats from Chongqing Municipality, China

Toxoplasmosis and anaplasmosis are severe zoonotic diseases, the former caused by Toxoplasma gondii and the latter by Anaplasma spp. In the present study, 332 goat blood samples were randomly collected from Chongqing Municipality, China to screen for T. gondii and Anaplasma spp. We used a polymerase chain reaction (PCR) to detect DNA, and enzyme-linked immunosorbent assay (ELISA) to test for T. gondii antibodies. The prevalence of T. gondii and Anaplasma spp. was 38% and 35% respectively by PCR, and 42% for T. gondii antibodies by ELISA. The co-infection rate by T. gondii and Anaplasma was 13%, where the two predominant pathogens co-infecting were Anaplasma phagocytophilum + A. bovis (10%), followed by T. gondii + A. phagocytophilum (9.64%). While co-infection by three pathogens varied ranging from 1.81% to 5.72%, less than 1% of goats were found to be positive for four pathogens. This is the first investigation of T. gondii and Anaplasma spp. infection in goats from Chongqing.


Introduction
Protozoan parasites and tick-borne infectious pathogens are common threats to both humans and animals [8,30]. The causative agent of toxoplasmosis, Toxoplasma gondii, is an obligate apicomplexan intracellular protozoan that can cause behavioral changes, neuropsychiatric disorders, abortions, stillbirth or fetal malformations, infertility and even death in humans and other mammals [19,21,24,26]. Anaplasmosis is caused by Anaplasma, a tick-borne pathogen that leads to inappetence, progressive anemia, fever, weight loss, milk production decrease, abortion, and sometimes death [14,18,25,34]. Infection by T. gondii and Anaplasma in goats not only affects the economic development of the animal industry, but can also have serious effects on human health [6,7,23]. Several surveys of T. gondii infection [20,31,32,33,40,41] or Anaplasma infection [18,[36][37][38] in goats have been reported in some regions of China. However, they all focus only on T. gondii or Anaplasma infection; none examine co-infection by these pathogens. The presence of A. phagocytophilum can alter the immune system of the host and make the animal more susceptible to other parasitic agents [22]. It is important to study the relevance of this phenomenon regarding T. gondii and Anaplasma spp. infection in goats in Chongqing.
Chongqing Municipality is located in southwest China and has been incorporated into the national "Advantage of agricultural products regional planning". It is recognized as a key area for goat breeding in China. However, there are no data on the prevalence of T. gondii and Anaplasma spp. infections in goats in Chongqing. The objective of this study was to investigate the prevalence of T. gondii, Anaplasma spp. and co-infection in goats in Chongqing, through detection of relevant pathogen DNA by PCR, and detection of T. gondii antibodies by enzyme-linked immunosorbent assay (ELISA).

Materials and methods
Collection of blood samples and DNA extraction The blood samples were collected from 332 apparently healthy goats randomly selected from 19 farms in 9 counties (Jiangjin, Dazu, Fuling, Rongchang, Tongnan, Youyang, Xiushan, Yunyang, and Zhongxian) of Chongqing ( Fig. 1), from October 2014 to April 2016. The breeds of goats included the Boer goat, Dazu black goat, Chengdu ma goat, Nanjiang yellow goat, and Hybrid black goat. The sera were stored at À20°C for T. gondii antibodies detection, and the blood samples were used for genomic The amplified PCR products were separated by electrophoresis in 1.5% agarose gels. The primers and amplification conditions are listed in Table 1.

Detection of T. gondii antibodies by ELISA
Serum antibodies against T. gondii were screened using the IDEXX Toxotest ELISA kit (IDEXX Laboratory, Westbrook, ME, USA), according to the manufacturer's recommendations. The serum samples and controls were diluted to 1:400 and tested in duplicate. The optical density (OD) was measured at 450 nm with an ELISA plate reader (Thermo Fisher, Waltham, MA, USA). The S/P (samples/positive control) percent for each sample was calculated according to the formula: S/P% = (OD 450 of the sample À OD 450 of negative control)/(OD 450 of positive control À OD 450 of negative control) Â 100. S/P% of samples less than 20 were considered negative for T. gondii antibodies. Samples with S/P% between 20 and 30 were considered questionable. If the S/P percentage was higher than or equal to 30, the samples were considered positive. If a sample remained suspect after a second run, a new sample from the same animal was collected and analyzed again. If the test result was again suspect, this sample was considered positive for T. gondii antibodies.

Statistical analysis
The prevalence of T. gondii and Anaplasma infection in goats of different sexes and ages was analyzed using the Chi Square Test in SPSS (version 18.0, SPSS Inc., Chicago, IL, USA), and the probability (p) value of < 0.05 was considered statistically significant.

Results and discussion
The molecular prevalence of T. gondii was estimated to be 37.65% population, and the seroprevalence was 42.47% by ELISA ( Table 2). The prevalence of T. gondii in goats has been reported to vary from 1.34% to 55.18% [1,2,4,11,12,29]. The relatively high prevalence of T. gondii in goats in Chongqing may be related to: 1) the oocysts of T. gondii excreted by infected cats that can easily develop to infective stages under the subtropical monsoon climate and humid weather in Chongqing and that are ingested by goats during grazing, and 2) the fact that most goats investigated in Chongqing were semihoused, potentially increasing the risk of T. gondii sporulated oocyst ingestion in wild grazing conditions. The prevalence of T. gondii in goats in Chongqing Municipality was obviously higher than that of goats in other places in China, with the prevalence varying from 3.8% to 14.1% [16,20,31,32,40]. Similar to a previous report [31], the prevalence of T. gondii in female goats (39.91% by PCR and 44.96% by ELISA) in Chongqing was higher than that of males (31.91% by PCR and 36.17% by ELISA), and goats aged 1 year or more were more highly infected than those less than one year old. The overall prevalence of Anaplasma infection in goats in Chongqing was 34.94% (Table 2), which was comparable to Anaplasma infection in Yunnan and Henan provinces (36.5%) [39], but higher than rates reported by other investigators for goats [36,37] and lower than rates for goats from Henan, Guizhou, Zhejiang and Hubei provinces in China [18]. Contrary to the prevalence of T. gondii in goats by sex, the prevalence of Anaplasma (37.23%) was higher in males than in females (34.03%). The prevalence of Anaplasma in goats aged one year or more (37.96%) was higher than that in goats less than 1 year old (29.31%). This is consistent with other reports [5,15]. The difference could be due to the fact that older animals are exposed to several tick seasons [5] and have a greater chance of exposure to ticks carrying Anaplasma spp. [15]. Similar to a previous study [37], 22.89% (76/332) of goats were positive for A. phagocytophilum infection followed by A. bovis (62/332, 18.67%) and A. ovis (43/332, 12.95%), which was not consistent with other reports indicating that the prevalence of A. phagocytophilum in goats was lower than that of A. bovis and A. ovis [18]. In addition, the prevalence of A. phagocytophilum in goats in this study was higher than that of goats in Slovakia [8]. Unlike a previous report [18], the dominant co-infection of A. phagocytophilum + A. bovis (34/332, 10.24%) was higher than A. phagocytophilum + A. ovis (22/332, 6.63%) and A. bovis + A. ovis (16/ 332, 4.82%). Co-infection by three Anaplasma spp. occurred in only 2.11% of the goats studied, which is similar to the other report [18] (Fig. 2). Co-infection by T. gondii and Anaplasma has been reported in rodents [27], dogs [10], ticks [35], and wild boars [22]. However, a survey on the occurrence of goats co-infected by T. gondii and Anaplasma was only reported in Slovakia [8]. In this study, 43 out of 332 (12.95%) goats were positive for T. gondii and Anaplasma (Fig. 2), indicating a relatively high prevalence of these two pathogens. The dominant co-infection between T. gondii and a single Anaplasma species was T. gondii + A. phagocytophilum (9.64%, 32/332), followed by T. gondii + A. bovis (8.43%, 28/332) and T. gondii + A. ovis (4.22%, 14/332). The high prevalence of A. phagocytophilum and T. gondii co-infection confirms the hypothesis that the presence of A. phagocytophilum can alter the immune system of the host and make the animal more susceptible to other parasitic agents [22]. A. bovis was first reported to infect goats in China by Liu et al. (2012). The high prevalence of A. bovis in this study also confirmed that goats may be an important natural reservoir for this organism [18]. Three-pathogen coinfection by A. ovis + A. phagocytophilum + T. gondii, A. phagocytophilum + A. bovis + T. gondii, and A. bovis + A. ovis + T. gondii was 2.71% (9/332), 5.72% (19/332) and 1.81% (6/332) respectively. Four-pathogen co-infection (A. ovis, A. bovis, A. phagocytophilum and T. gondii) was simultaneously detected in 3 goats (0.9%) (Fig. 2).
The main species of tick in Chongqing is Boophilus microplus [28], which is one of the vectors of Anaplasma phagocytophilum in China [37]. Since T. gondii has already been detected in ticks [35], a study of T. gondii and Anaplasma carriage by Boophilus microplus in Chongqing should be carried out.