Abstract
Toxoplasma gondii infections are prevalent in humans and animals worldwide. The aim of the study was to estimate the seroprevalence of T. gondii in pet rabbits, as well the presence of T. gondii DNA in their blood. A total of 360 pet rabbits were investigated for the presence of antibodies and antigens of T. gondii in blood samples using a modified agglutination test (MAT) and nested PCR, respectively. Antibodies against T. gondii were found in 44 (12.12%) of pet rabbits. In rabbits that received unwashed vegetables 13.13% were positive for T. gondii antibodies, whereas all rabbits that received washed vegetables were seronegative. The prevalence of anti-T. gondii antibodies was statistically higher in samples collected from ill rabbits (45.45%) compared with healthy ones (8.87%), as well in the group of rabbits that had contact with cats (16.13%) compared with those without contact with cats (3.57%). The percentage of seropositive samples derived from all three sampling regions of Poland was as follows: Silesia (12.20%), Lower Silesia (6.09%), and Lesser Poland (18.03%). No statistical differences in seroprevalence were observed according to age or sex of rabbits. All tested blood samples were negative for the T. gondii B1 gene in nested PCR. This is the first study of seroprevalence and the presence of T. gondii in the blood of pet rabbits in Poland. Our study indicates that health status of rabbits, contact with cats, as well sampling region may have an important impact on the prevalence of T. gondii infection.
Introduction
Toxoplasmosis is one of the most common parasitic infections in humans and in domesticated and wild animals all over the world. Toxoplasma gondii is an obligate intracellular protozoon that infects all warm-blooded vertebrates, resulting in neuromuscular diseases and abortion in different species (Dubey 2010). Rabbits are one of the intermediate hosts, whereas felines are the only known definitive hosts of T. gondii. The cause of infection in rabbits is from drinking water or consumption of food contaminated with sporulated oocysts from cat feces or congenital transmission from the rabbit dam to the fetus (do Nascimento et al. 2017). Domestic and meat-producing rabbits with latent T. gondii infections are considered to be a potential source of human toxoplasmosis (Ishikawa et al. 1990, Sroka et al. 2003).
The main route for humans to acquire T. gondii infection is the consumption of raw or undercooked meat, included rabbit meat, or by consumption of water, vegetables or fruits contaminated with oocysts, as well by transplantation, blood transfusion, and congenitally (Dubey and Jones 2008, Dubey 2010, Lass et al. 2012). Therefore, the European Food Safety Authority (EFSA) has recommended the surveillance and monitoring of toxoplasmosis in humans, animals, and foodstuffs (EFSA and ECDC 2016). Contamination of the environment with oocysts shed by definitive hosts is considered a potential risk both for humans and other animals (Dubey and Jones 2008). Because many pet rabbits have contact with cats at home, many of them eating a lot of unwashed vegetables, fruits, sprouts, leaves, and so on, the possibility of transmission seems to be quite high.
In most cases T. gondii infection in rabbits is not manifested in any clinical symptoms. After the primary infection, the parasite can stow away in a latent state in nervous tissues and muscles for long periods of time, even as long as the animal lives. Such subclinically infected animals carry circulating antibodies that can be a sign in different serological tests. However, the parasite may manifest itself clinically in abortions or neuromuscular symptoms such as pyrexia, lethargy, paresis, posterior paralysis, head tremor, and ataxia.
There is a large amount of research data about the prevalence of T. gondii in wild, farm, domestic, or pet rabbits worldwide (Almería et al. 2004, Sroka et al. 2007, Alvarado-Esquivel et al. 2013, Neumayerová et al. 2014, Salman et al. 2014, Mason et al. 2015, Meng et al. 2015, de Lima et al. 2016, Wang et al. 2018). However, no data are available on the prevalence of T. gondii in Polish pet rabbits. To our knowledge, this is the first study of seroprevalence and DNA detection of T. gondii in pet rabbits in Poland.
Materials and Methods
Sampling
Blood samples from 360 pet rabbits were collected between December 2017 and May 2018. There were 184 male and 176 female pet rabbits, which came from three regions in Poland (Silesia, Lower Silesia, and Lesser Poland). The general characteristics of the studied rabbits are summarized in Table 1.
General Characteristics and Prevalence of Toxoplasma gondii in Pet Rabbits in Poland (n = 360)
Two separate samples of blood (ca. 1 mL per sample) were collected from one rabbit—the first one (w/o EDTA) dedicated to the serum, and the second one with EDTA for nested-PCR. Blood samples were obtained from the central ear artery (lat. arteria auricularis centralis) or from the saphenous vein (lat. vena saphena) during routine veterinary clinical examination. The whole blood samples (w/o EDTA) were centrifuged at 3000 g for 10 min. to obtain sera for the modified agglutination test (MAT). All samples were frozen and stored at the temperature range of −20°C to −25°C until further examinations.
For all rabbits studied a questionnaire about feeding rules (feeding with washed or unwashed vegetables), contact with cats (current or in the past, in the aspect of possible contact with oocysts), and health status (the influence of health status of pet rabbit to T. gondii infection) was prepared. Data collected through these questionnaires were subjected to analysis of the risk factors.
Modified agglutination test
Each rabbit's serum sample was tested for the presence of T. gondii antibodies by the MAT as described previously (Dubey and Desmonts 1987) using a commercial kit (ToxoScreen DA, bioMérieux, Lyon, France). Positive and negative controls were based on formalin-fixed tachyzoites as antigens. The T. gondii antigen and rabbit positive control sera were provided by MyBioSource, Inc. (San Diego, CA). Samples were considered positive when agglutination was observed at a dilution of 1:25 or higher (Almería et al. 2004, Alvarado-Esquivel et al. 2013).
DNA extraction
Genetic material from blood was isolated using Xpure™ Blood Mini (A&A Biotechnology, Gdańsk, Poland) according to the manufacturer's recommendations. DNA was quantified spectrophotometrically (BioPhotometer, Eppendorf, Hamburg, Germany) and stored at the temperature range of −20°C to −25°C.
Nested PCR was performed using two primer pairs (Genomed, Warsaw, Poland), to amplify the B1 gene, as described by Grigg and Boothroyd (2001) as follows: S1 (5′-TGTTCTGTCCTATCGCAACG-3′) and AS1 (5′-ACGGATGCAGTTCCTTTCTG-3′), which amplify a 580-bp fragment, and S2 (5′-TCTTCCCAGACGTGGATTTC-3′) and AS2 (5′-CTCGACAATACGCTGCTTGA3′), which amplify a 530-bp fragment. Amplification was performed according to the conditions described by Alfonso et al. (2009). Products obtained by amplification were divided using electrophoresis in 2% agarose gel. DNA bands were stained with Midori Green DNA Stain (Nippon Genetics Europe GmbH, Dueren, Germany) and visualized with a UV transilluminator.
Statistical analysis
Statistical analysis was performed by Fisher's exact test using the PQStat Statistical Program version 1.6.2 (PQStat, Poznań, Poland). Seroprevalence was statistically analyzed considering the age, sex, and regions of sampling, food, contact with cats, and health status of the animals. A p value of ≤0.05 was considered statistically significant.
Results
Antibodies (MAT ≥1:25) to T. gondii were found in 44 (12.22%) of the 360 pet rabbits, derived from all three sampling regions: Silesia, Lower Silesia, and Lesser Poland. A statistically higher seroprevalence of T. gondii antibodies was observed in ill pet rabbits (15; 45.45%) in comparison with the healthy ones (29; 8.87%) (p < 0.001). In the group of rabbits that had contact with cats, a statistically significant (p < 0.001) higher percentage of seropositive animals was observed (16.13%). In the group of rabbits that had no contact with cats only four (3.57%) animals were positive in anti-T. gondii antibodies. Similar results were found in rabbits that received unwashed vegetables: 13.13% of animals were positive for anti-T. gondii antibodies. In total, only 6.94% of all responding owners of rabbits declared that they washed vegetables. Regarding the sampling region, 12.20% of samples collected in Silesia were positive for anti-T. gondii antibodies, in Lesser Poland it was 18.03% and in Lower Silesia only 6.09%. No statistically significant differences were observed in the prevalence of anti-T. gondii antibodies for sex or age of rabbits. All results and data collected from the questionnaires are included in Table 1.
Discussion
The population of pet rabbits in Poland has increased in recent years, while keeping rabbits as companion animals has become very popular. In our study, we focused on the seroprevalence of T. gondii, as well on detection of T. gondii DNA in blood sera in pet rabbits. In this study, a 12.22% seroprevalence of T. gondii in pet rabbits was detected, which is in agreement with results obtained by other authors (Almería et al. 2004, Alvarado-Esquivel et al. 2013, Shin et al. 2013, Wang et al. 2018). Almería et al. (2004) and Shin et al. (2013) reported that 14.2% of wild rabbits in Spain and 10.6% of farm rabbits in Korea, respectively, possessed anti-T. gondii antibodies. In the study by Wang et al. (2018) in China, 10.55% of domestic rabbits were positive for anti-T. gondii antibodies using the MAT. Alvarado-Esquivel et al. (2013) found antibodies to T. gondii in 16.3% of domestic rabbits from backyards, farms, and pet shops in Mexico. Research into the seroprevalence of T. gondii in rabbits in Poland was conducted by Sroka et al. (2003, 2007), but only in a group of farm rabbits. The presence of anti-T. gondii antibodies was found in 22.2% and in 7.4% of farm rabbits in 2003 and 2007, respectively. Both these studies were performed in Lublin province. In our study, the samples were collected in different regions of Poland and the seroprevalence was as follows: in Silesia (12.20%), Lower Silesia (6.09%), and Lesser Poland (18.03%).
In this study, pet rabbits fed with unwashed vegetables showed a higher seroprevalence of T. gondii infection (13.13%) than the pet rabbits fed with washed vegetables (no seropositive animals). Alvarado-Esquivel et al. (2013) also observed the highest seroprevalence of T. gondii antibodies in rabbits fed with a mixture of concentrated food, fruits, vegetables, and grains. In Poland, Lass et al. (2009) detected the presence of T. gondii oocysts on fresh fruits and vegetables from shops and gardens, suggesting environmental contamination. Moreover, these authors detected the DNA of T. gondii by real-time PCR in 8% of the food samples bought in shops and bazaars, whereas in samples taken from gardens it was 17.07%. The results obtained in our study, as well by other authors may suggest that unwashed vegetables contaminated by T. gondii oocysts may be a source of infection not only in animals but also in humans.
Regarding contact of pet rabbits with cats, in our research, the animals that had contact with cats were positive in T. gondii antibodies in 16.13%, whereas pet rabbits that had no contact with cats only in 3.57%. This result may indicate on the potential role of domestic cats in spreading of T. gondii infection by shedding oocyst to the environment without any clinical symptoms of the disease, especially in the aspect that rabbits and cats that are kept at home together, they usually have close contact, they play or rest in the same place.
In this study, the seroprevalence of T. gondii in pet rabbits was not related to sex or age, which is consistent with results obtained by Almería et al. (2004). In our study, the main factor affecting the prevalence of infection was the health status of rabbits. Human toxoplasmosis is present especially in individuals with an impaired immune system (Hill and Dubey 2002, Saadatnia and Golkar 2012). In our opinion, ill rabbits may have been immunosuppressed and, therefore, may become more susceptible to other infections, for example, to toxoplasmosis.
In our study, all tested blood samples were negative for the presence of T. gondii in nested PCR. Shin et al. (2013) revealed that 16.2% of blood samples obtained from healthy farm rabbits in Korea were positive for T. gondii, whereas de Lima et al. (2016) observed the presence of T. gondii DNA in the tissue fragments of 9.25% of domestic rabbits. In our study, lack of positive results in nested PCR among seropositive rabbits was most likely related to T. gondii infection in pet rabbits in the past. Moreover, the difference between the results obtained by different authors may arise from the fact that T. gondii is temporarily present in blood and, therefore, it is possible that it is not readily detected by nested PCR.
Conclusions
To the best of the authors' knowledge, this is the first study focused on T. gondii seroprevalence and DNA detection in pet rabbits from Poland. The obtained results revealed that the health status of pet rabbits, contact with cats, as well as eating unwashed vegetables may be a possible risk factors of T. gondii infection.
Footnotes
Acknowledgments
We gratefully thank Sebastian Ploch from IT laboratory, faculty of veterinary medicine, Wroclaw University of Environmental and Life Sciences, for statistics analysis of obtained data.
Author Disclosure Statement
No competing financial interests exist.
Funding Information
No funding was received for this article.