Cross-Sectional Serosurvey and Risk Factors Associated with the Presence of Toxoplasma gondii Antibodies in Pigs in Greece

Introduction

Toxoplasmosis is a zoonotic disease with worldwide distribution caused by the protozoan Toxoplasma gondii. Cats are definitive hosts shedding infective oocysts in their feces. Pigs may become infected by ingesting feed or water contaminated with cat feces, by cannibalism of other infected dead pigs, by ear and tail biting or by eating infected rodents or other uncooked meat (Dubey 2009a, Tenter et al. 2000).

Postnatally acquired infections are generally asymptomatic and self-limiting, despite the persistence of the parasite in the edible tissues of pigs, even for life (Dubey 2010). However, ingestion of infected porcine meat containing tissue cysts is considered as the major meat source of Toxoplasma infection in humans (CDC 2015, Schluter et al. 2014). Even though a decreasing rate of human cases has been reported in Europe, 40 confirmed congenital toxoplasmosis cases were reported by 19 European Union member states in 2012, and toxoplasmosis still remains one of the most prevalent parasitic zoonotic diseases (ECDC 2014). Similarly, the presence of antibodies against T. gondii in a high percentage (11.1%) of children in northern Greece has been reported (Frydas et al. 2000). Furthermore, an incidence rate of 0.1 cases per 10,000 births per year (from 2007 to 2009) was reported by a dedicated surveillance network for congenital toxoplasmosis in Greece (Aptouramani et al. 2012). In a review on the global status of T. gondii, average seroprevalence of toxoplasmosis in pregnant and childbearing women ranged between 20.1% and 29.4% and 20% and 36.4% respectively (Pappas et al. 2009). Toxoplasmosis is usually a mild nonspecific or asymptomatic infection; therefore, antibody detection has been used to support diagnosis, mostly in cases of reproductive failure in pregnant females and hence, usually to detect subclinical infections (Dubey 2010).

Serological testing is the most commonly used technique for identifying exposure to T. gondii in humans and animals alike. Several serological tests with diverse reliability have been used for the detection of T. gondii antibodies in pigs (Dubey 2009b), such as Sabin–Feldman dye test, complement fixation test, indirect hemagglutination, latex agglutination test, modified agglutination test, Western blot, indirect fluorescent antibody test (IFAT), and enzyme-linked immunosorbent assay (ELISA) (Dubey 2009b, Steinparzer et al. 2015). IFAT has been widely used for the detection and mainly quantitation of T. gondii–specific immunoglobulin G (IgG) and IgM antibodies, while ELISA is well suited to laboratories required to analyze large numbers of samples (OIE 2008).

Antibodies against T. gondii have been found in pigs worldwide (Dubey 2009b). Seropositive pigs could harbor tissue cysts in their meat thereby representing a tentative risk for porcine meat consumers (Tenter et al. 2000, Dubey 2010). To the authors' best knowledge, the seroepidemiology of toxoplasmosis in pigs is largely unknown in Greece. Therefore, this study was carried out to assess the serological status of pigs from different farms, employing both IFAT and ELISA, and to identify the factors associated with the risk of infection. In order to investigate the risk factors for seropositivity in pig herds and to identify high-risk farms, the study focused on environmental risks, the farm size and biosecurity level in pig herds under various farming conditions in Greek mainland.

Materials and Methods

Sera (n = 609) from pigs living in 65 pig farms in Greek mainland were used in the study. Blood samples were collected from 10% of the sows of each farm. Sera were separated from whole blood after centrifugation and kept frozen pending analysis. The only criterion for the selection of the farms was the willingness of the farmer to participate in the study. Information about the geographical location, size, and biosecurity level of the herd were recorded. According to the geographical data, the farms located in the mountains were classified as mountainous and those in the plains as lowland farms. The categories for the farm size were small (≤100 sows), medium (101–300 sows) and large (>301 sows). Herd-level information was recorded in the data capture form presented in Table 1. The information concerned various aspects of herd management, husbandry, biosecurity measures, and the environment. It was hypothesized that these variables could be related to the spread of the infection to pigs within herds. A face-to-face interview of the farmer was followed by a pig herd examination to assess the clarity of questions and confirm the validity of information on-site. All herds were visited once by the same investigator to avoid any observer variation while filling in data capture forms. Given answers along with inspection results of actual conditions were combined by the investigator to fill in the data capture form. The biosecurity level of the farm was characterized as poor, good, and very good based on the predefined classifications as presented in Table 1.

An ELISA test was performed using a commercial test kit (multi-species ID Screen^® Toxoplasmosis Indirect, IDVET, Montpellier, France) for the detection of serum antibodies against the Toxoplasma P30 protein as per manufacturer's instructions.

Anti-T. gondii antibodies were also detected by indirect fluorescence antibody test kit using commercially available slides coated with parasite tachyzoites (Fuller Laboratories, Fullerton CA) and anti- porcine IgG conjugate (Porcine IgG FITC conjugate, VMRD Inc., Pullman, WA) was used. Serum samples were tested at two-fold dilutions in phosphate buffered saline (PBS) as previously described (Edelhofer 1994, Veronesi et al. 2011), until reaching the endpoint titer. Farms with at least one positive sample were recorded as positives, and those with no positive sample were characterized as negatives. The information recorded at each farm was transferred to SPSS^® v. 21 for statistical analysis. The categorical variables herd size, biosecurity level, and geographic characteristics of the herd were tested as potential risk factors for the presence of toxoplasmosis in the herd with logistic regression analysis. Initially, the main effects of all tested risk factors were evaluated and then the model was run stepwise forward with probability for entry in the model p < 0.05 and for removal p > 0.1. The last category of each variable was used as the reference variable. Chi-squared was run to determine the significance of the difference of positive herds' percentages among the levels of all categorical parameters.

The kappa (κ) agreement between ELISA and IFAT was calculated using Graph Pad Prism v.6 (Graph Pad Inc., San Diego, CA).

Results

In total, 609 blood samples were tested for toxoplasmosis. Twenty-six positive samples were detected (4.3%) in 17 out of the 65 farms tested (26.2%). The average seroprevalence in affected herds was 26.8% (95% confidence interval: 21.0–32.6%). The comparative results of ELISA and IFAT for the detection of anti-Toxoplasma antibodies are presented in Table 2.

A very good agreement was observed between the two employed serological methods with a k-value of 0.958 (95% confidence interval: 0.901–1.000);

The frequency of seropositivity against T. gondii according to the geographical relief, farm size, and biosecurity level is presented in Table 3. Among the categorical data evaluated, the biosecurity level (p < 0.01) and the geographical characteristics (p < 0.05) were identified as independent risk factors for the presence of toxoplasmosis in the herd (Table 3). Farms in lowlands and those with very good biosecurity level were at lower risk for toxoplasmosis compared with mountainous farms and those with poor and good biosecurity level, respectively (Table 4). Additionally, Toxoplasma infection was recorded in higher percentages in small and medium sized than in large farms, but this difference was not significant.

Discussion

The results of this study support the assumption that pigs are exposed to T. gondii in Greece. This is the first cross-sectional seroepidemiologic study in pigs in Greece, although the presence of antibodies against T. gondii has been reported in humans (Decavalas et al. 1990, Diza et al. 2005,(Bobic et al. 2011) and other species such as cats (Chatzis et al. 2014), small ruminants (Diakoua et al. 2013) and horses (Kouam et al. 2010) in this country.

In the present study both ELISA and IFAT were used. Our study showed almost perfect agreement between these two different methods for antibody detection, which suggests they can be used interchangeably. Although the use of k-value for method comparison has the limitation of being dependent on prevalence, the very high k-value (0.958) obtained in our study is considered to be adequate from a test substitution perspective. Therefore, the IFA test can be replaced by ELISA, which might be preferable based on cost and sample through put capacity (Gardner et al. 2010).

Serologic surveys have documented exposure of pigs to T. gondii worldwide (Tenter et al. 2000, Dubey 2009b). In southern Europe, antibodies against T. gondii were detected in Serbia (Klun et al. 2006, Klun et al. 2011) in Italy (Genchi et al. 1991, Veronesi et al. 2011) and Spain (Garcia-Bocanegra et al. 2010a, Garcia-Bocanegra et al. 2010b). T. gondii seroprevalence in pigs varies from less than 1% to 90.4% among different countries (Dubey, 2009b). Even though accurate comparisons with the results of studies in other countries cannot be made since the size of sample, the population sampled, the method of analysis and the cut-off of the method are not the same, it seems that seroprevalence in pigs in Greece is comparatively moderate.

Of note, the present study sampled only sows, in which a higher seroprevalence than in finishing pigs has usually been reported. (Dubey et al. 1991, Dubey et al. 2005).

Considering biosecurity, summarized data on toxoplasmosis in pigs from the United States revealed a decrease in seroprevalence during a decade, which was more prominent in well managed facilities (Dubey and Jones 2008) To the contrary an increase of seroprevelance was reported in outdoor reared pigs in the Netherlands (van der Giessen et al. 2007). In the present study, all animals were kept indoors; therefore, this factor cannot be evaluated.

The concept of biosecurity briefly regards the avoidance of disease or further spread of the disease. According to the International Organization of Epizooties (OIE) biosecurity is defined as the implementation of measures that reduce the risk of the introduction and spread of disease agents; it requires the adoption of a set of attitudes and behaviors by people to reduce risk in all activities involving domestic, captive/exotic and wild animals and their products (OIE 2010).

From the definition it becomes obvious that biosecurity measures vary a lot depending on the farm size and production system as well as social, economic, and cultural factors. For these reasons biosecurity measures that to the authors' knowledge are applied in the Greek pig industry are presented and classified in Table 1.

Analysis of risk factors in the present study showed that farms with very good biosecurity measures presented the lowest risk of being infected by Toxoplasma. In contrast, the farms with poor or good biosecurity had 50- and 23-times higher risk for toxoplasma infection respectively. Among the criteria for farm classification according to biosecurity level was the application of a protocol to keep out rodents. The role of rodents in the life cycle of Toxoplasma is well known and there are strong indications that they act as reservoirs for direct transmission of T. gondii. Implementation of rodent control campaign significantly reduced seroprevalence in organic farms (Kijlstra et al. 2008). Similarly, cats, the definite hosts of T. gondii and the more obvious risk factors as reported in previous studies (Dubey et al. 1997, Tao et al. 2011), were excluded from farms with very good biosecurity measures, therefore reducing the risk of pig infection. A high number of cats were positively correlated to T. gondii seroprevalence in pigs (Meerburg et al. 2006).

Biosecurity measures also prevent contact between pigs and wildlife, which is another source of T. gondii (Ferroglio et al. 2014, Touloudi et al. 2015). The seropositivity rate in pigs in our study was much lower than the rate reported in pregnant women (Pappas et al. 2009). Apparently, although tissue cysts are frequently observed in infected pigs, other meat-producing animals such as sheep and goats, and less frequently poultry and rabbits, might have been the source of infection in pregnant women in that study. Furthermore, human infection is also possible by food or water contaminated with feline feces (Tenter et al. 2000).

In pigs, altitude was found to be a significant risk factor, being positively associated with seroprevalence that increases in mountainous areas and decreases in the lowlands. Altitude has been reported as a risk factor associated with toxoplasmosis in pigs and is related to environmental conditions, proximity to possible sources of infection and different management strategies (Villari et al. 2009).

On the other hand, farm size was not associated with the infection, although large farms presented the lowest prevalence, probably due to differences in management systems, since large farms in Greece employ strict hygienic measures that prevent T. gondii oocysts from spreading throughout their facilities. Conversely, medium and small size farms, run as family businesses, may have inadequate hygienic standards that allow the consequent spread of T. gondii oocysts to their animals. Similar results have been previously reported and were attributed to more frequent outdoor rearing of pigs at smaller rather than at large swine farms (Assadi-Rad et al. 1995).

Based on the present study, it is reasonable to assume that pigs raised enclosed at all times in environmentally regulated buildings in the absence of contact with sources of infection such as animal hosts of the parasite or passive oocyst transfer through contaminated workers' footwear, are least likely to have T. gondii. Knowledge of the relationship between the seropositivity against T. gondii and sow herd management/environmental factors would be useful in the development of improved control strategies.