Biologia 65/4: 670—674, 2010 Section Zoology DOI: 10.2478/s11756-010-0070-2

Prevalence estimation and genotypization of Toxoplasma gondii in goats

František Spišák, Ľudmila Turčeková, Katarína Reiterová, Silvia Špilovská &PavolDubinský

Parasitological Institute, Slovak Academy of Sciences, Hlinkova 3, 040 01 Košice, Slovak Republic; e-mail: [email protected]

Abstract: In this study we aimed to determine seroprevalence of antibodies against Toxoplasma gondii and parasite DNA presence in the milk of goats from a farm in eastern Slovakia. Anti-Toxoplasma antibodies were detected in 43 goat sera out of 87 examined (49.43%). The highest prevalence was recorded in the goats aged more than 72 months (76.19%; OR = 4.62; 95% CI = 1.51–14.14) and the lowest one in animals aged from 12 to 36 months (17.65%; OR = 0.09; 95% CI = 0.03–0.27). Statistically significant correlation (P < 0.0001) was found between the prevalence of antibodies against T. gondi and animal age in comparing age groups – goats up to 36 months of age and above 37 months of age. The presence of T. gondii DNA was confirmed in 32.56% of milk samples using molecular methods. Based on the DNA polymorphism at the SAG2 locus of T. gondii we identified the goats as being infected with genotype II of T. gondii.PresenceofDNAin milk refers to the risk of human infection through consuming raw milk. Key words: Toxoplasma gondii; seroprevalence; milk; goats; genotype; Slovakia

Introduction and milk (Neto et al. 2008). Infected goats represent an important source of Toxoplasma infection in Islamic Toxoplasma gondii is the facultative two-host, tissue where consumption of unpasteurized goat cyst-forming coccidium, which is prevalent in most ar- milk due to their cultural traditions (Jittapalapong et eas of the world. The definitive hosts of T. gondii are al. 2005). domestic and wild carnivores of the family Felidae, and According to Figueiredo et al. (2001) drinking and the intermediate hosts are all warm-blooded animals control of raw goat milk is extremely important for the (mammals and birds) and humans. disease control and in particular for public health, since There are several modes of human infection with the consumption of goat milk is elevated in children T. gondii. The most frequent is through consumption of with allergy to cow milk. raw or undercooked meat containing viable tissue cysts The collection of present data about T. gondii is or ingestion of food or water contaminated with infec- of medical importance as a major source of parasitic tious oocysts from the faeces of infected cats. Though zoonosis (Fusco et al. 2007). Surveillance and moni- congenital transplacental transmission of tachyzoites toring of toxoplasmosis in humans and animals is per- is rarely seen, it is extremely harmful for the foetus formed regularly only in few countries, mainly within (Dubey 1996). According to Tenter et al. (2000), un- the European Union (EU). pasteurised milk may be an important source of hu- We aimed to determine the prevalence of toxoplas- man Toxoplasma infection. The highest risk of human mosis in goat sera from the farm in eastern Slovakia, to T. gondii infection is through ingestion of raw goat milk confirm the presence of T. gondii in the milk of seropos- (Sacks et al. 1982; Skinner et al. 1990). itive goats and to genetically characterize the agent of Toxoplasmosis in animals may remain clinically in- toxoplasmosis, determining its genotype. apparent, causing foetal resorption, foetal mummifica- tion, abortion at any stage of pregnancy and/or still- birth or birth of live but weak offspring in sheep and Material and methods goats (Masala et al. 2003). Toxoplasma infection has a significant impact on animal reproduction, both causing A total of 87 adult dairy goats were examined for antibod- ies against T. gondii using ELISA. Blood samples were col- economic losses, and jeopardizing public health since lected from the jugular vein, approximately two months af- consumption of infected meat or milk can facilitate ter delivery. The goats originated from the farm located in zoonotic transmission (Jittapalapong et al. 2005). Košice surrounding in eastern Slovakia, with a total Goats are economically important animals in many of 104 goats being bred, of which 87 were adults suitable countries and are the most important source of meat for our experiments. In the farm proper sanitary conditions

c 2010 Institute of Zoology, Slovak Academy of Sciences Toxoplasma gondii genotypization in goats 671

Table 1. Prevalence of Toxoplasma gondii in blood samples of goats according to their age.

ELISA Age (months) Number of Positive (%) OR 95% CI examined samples Negative and doubtful Positive Strong positive

12–36 34 28 2 4 17.65 0.09 0.03–0.27*+ 37–72 32 11 5 16 65.63 2.86 1.16–7.10* >72 21 5 7 9 76.19 4.62 1.51–14.14+

Total 87 44 14 29 49.43

OR – Odds ratio; CI – Confidence interval; *P < 0.0001; +P < 0.0001. were imposed, however, the goats were regularly pastured on Nested PCR analysis of SAG2 locus grassland where they could be contaminated with T. gondii. Genotypization of DNA samples of T. gondii from goat The milk samples from 43 seropositive goats were used milk was examined using nested PCR SAG2 locus. Sam- for DNA examination. The samples of milk were collected ples were analyzed separately for the 5’ and 3’ ends of the for analyses five months after delivery. locus, according Howe et al. (1997). The 5’ end of the locus was amplified by nested PCR with the primers SAG2: F4 Serological analysis (5’GCTACCTCGAACAGGAACAC3’) and R4 (5’GCAT- The goat sera were separated after sedimentation of blood CAACAGTCTTCGTTGC3’) and a following amplification ◦ cells and supernatant was stored at –20 Cuntiltested. with the internal primers F (5’GAAATGTTTCAGGTTGC Serum samples were tested for the presence of IgG anti- TGC3’) and R2 (5’GCAAGAGCGAACTTGAACAC3’). bodies against T. gondii using a commercial indirect ELISA The 3‘ end of the locus was amplified using primers F3 kit (ID.VET, Montpellier, France) according to the manu- (5’TCTGTTCTCCGAAGTGACTCC3’) and R3 (5’TCAA facturer’s instructions. For each sample, the ratio of optical AGCGTGCATTATCGC3’) in the first amplification and densities (OD) of examined serum to mean OD of positive the internal primers F2 (5’ATTCTCATGCCTCCGCTTC3’) control was calculated as S/P%. and R (5’AACGTTTCACGAAGGCACAC3’) in the sec- ond reaction. PCR products were electrophoresed on a 1.5% S/P% = ODvalue of the sample/ODvalue of the positive control × 100. agarose gel stained with Gold View (SBS Genetech, China) and visualized under the UV lamp at 254 nm. Any samples with an S/P less than or equal to 40% were considered as negative, the samples with an S/P be- RFLP analysis tween 40% and 50% were considered as doubtful, the sam- For genotyping of T. gondii parasites found in samples of ples with an S/P greater than or equal to 50% and less than goat milk, amplification products of the SAG2 locus were 200% were considered as positive and the samples with an analysed by RFLP method. The amplified fragments were S/P greater than or equal to 200% were classified as strong digested with restriction endonucleases Sau3A I at 5’ end positive. and with Hha I at 3’ end of locus according to protocols of Promega (Madison, USA). The position of fragments vi- DNA isolation sualized on 1.2% agarose gel characterized genotype of T. DNA was isolated from 43 milk samples by commercially gondii according to Howe et al. (1997). available kit (QIAamp DNA Mini Kit; QIAGEN, GmbH., Hilden, Germany). The DNA was isolated according to the Statistical analysis manufacturer’s protocol and stored at –20 ◦C for further in- The statistical analyses were performed using Fisher’s exact  vestigation. test (GraphPad Prism 5) with corresponding odds ratio (OR) for the examined variables and 95% confidence inter- PCR analysis of TGR1E gene val (95% CI). The differences were considered statistically The isolated DNA were analysed by the PCR method with significant when P < 0.05. two primers of TGR1E gene using the 35-fold repetitive sequence as a target. The sequences of primers for ampli- Results fication were used according to Lamoril et al. (1996): T1 (5’ATGGTCCGGCCGGTGTATGATATGCGAT3’) and The antibodies against T. gondii were detected in the T2 (5’TCCCTACGTGGTGCCGCATTGCCT3’). The pos- itive fragment after amplification consisted of 191 bp. sera of 87 lactating goats divided into three age groups. The DNA extracted was amplified using the following Group 1 comprised animals from 12 up to 36 months of conditions: three minutes of DNA denaturation at 94 ◦C, age, group 2 goats from 37 up to 72 months of age and followed by 40 cycles of amplification – 30 seconds at 94 ◦C, group 3 comprised goats above the age of 72 months 30 seconds at 60 ◦C, and 1 minute at 72 ◦C; the last step of (Table 1). Out of a total of 87 examined sera, 42 sam- ◦ extension for 7 minutes at 72 C. ples were negative and 2 samples were classified as du- PCR products were electrophoresed on a 3% agarose bious. The prevalence of toxoplasmosis in the herd was gel stained with Gold View (SBS Genetech, China) and vi- 49.43%, i.e., 43 seropositive samples. Positive samples sualized under UV lamp at 254 nm. PCZ strain (RH-like) were classified as positive and strong positive samples strain (NRL for Toxoplasmosis, Prague, Czech Republic) served as the positive control, and distilled water was used according to the protocol described above. as the negative control in all PCR experiments. 100 bp lad- Anti-T. gondi antibodies were detected in all age der (Promega, Madison, USA) was used as the molecular groups, though prevailing in the group of strong posi- weight marker. tive samples (29 of 87 examined samples, i.e., 33.3%, 672 F. Spišák et al.

Table 2. Presence of Toxoplasma gondii DNA in goat milk examined by molecular methods.

Occurrence of T. gondii Age (months) No. of milk samples Positive by TGR1E Genotype SAG2 DNA in milk (%)

12–36 6 1 II 16.67 37–72 21 5 II 23.81 >72 16 8 II 50.00

Total 43 14 32.56 were strong positive). The highest seroprevalence of lence of 20.2% in goats, Šlosárková et al. (1999) de- 76.19% (OR = 4.62; 95% CI = 1.51–14.14) was deter- tected seroprevalence of 66% in a herd of Angora goats. mined in Group 3 (goats older than 72 months of age) Paul (1998) examined potential risk factors (e.g., drink- and the lowest one (17.65%) (OR = 0.09; 95% CI = ing untreated milk) for T. gondii infections in humans 0.03–0.27) in Group 1 (up to 36 months of age). The (P = 0.02) in Poland. Edelhofer & Prossinger (2010), differences among the age groups were statistically sig- who carried out seroepidemiological studies in Austria, nificant (P < 0.0001). The difference between Group 2 recorded the seroprevalence of 69%. The mean preva- (37 to 72 months of age) and Group 3 (over 72 months lence of 47% in goats was recorded in the Netherlands of age) was statistically insignificant. (Antonis et al. 1998). Only 15% prevalence of tox- The presence of T. gondii TGR1E gene was de- oplasmosis was reported from Switzerland (Chanton- tected in the milk samples collected from seropositive Greutman et al. 2002) but a lower prevalence (11.8%) animals and the parasite was genetically characterized was detected in small ruminants (sheep, goats) in Spain using SAG2-RFLP method. Molecular analysis con- by Mainar et al. (1996). Prelezov et al. (2008) detected firmed the presence of the parasite DNA in 14 milk 59.8% seroprevalence of toxoplasmosis in Bulgaria and samples, i.e., (32.56%). Presence of T. gondii DNA in Masala et al. (2003) recorded the prevalence of 12.3% milk correlated with seropositivity of animals (Table 1 in goat herds on Sardinia, Italy. and 2). ELISA test, performed on 87 sera samples of lac- In all the cases, T. gondii was characterized as tating goats, revealed 49.43% seroprevalence. Our re- genotype II (Table 2). sults are in concordance with those from the Nether- lands. Acute toxoplasmosis was detected in 29 goats, Discussion with the highest number of affected animals in Group 2 (37 to 72 months of age) (OR = 2.86; 95% CI = Consumption of raw food, especially among immuno- 1.16–7.10), while Group 1 (up to 36 months of age) compromised persons and pregnant women, poses a po- included only four infected animals. (OR = 0.09; 95% tential threat to public health. Especially goat milk rep- CI = 0.03–0.27) (Table 1). Total prevalence gradually resents a risk factor as it is usually consumed raw (un- increased from the youngest to the oldest age group, treated) (Fusco et al. 2007). with the prevalence rates 4.3 times higher in Group 3 Presence and survival of T. gondii tachyzoites in comparingtoGroup1(P < 0.0001). Similar correlation goat milk were studied by Walsh et al. (1999). They ex- between the prevalence rates and age has been observed perimentally confirmed that tachyzoites survived in the by Jittapalapong et al. (2005). Similarly, Cavalcante et goat milk for three to seven days at 4 ◦C, thus proving al. (2008) found that the goats aged 37 months and that raw goat milk can serve as a source of Toxoplasma more were at a higher risk of acquiring toxoplasmosis infection. Potential transmission of T. gondii from the than younger age groups (OR = 2.01; 95% CI = 1.55– goat milk was also reported by Halánová et al. (2008). 2.61). The higher prevalence rates of toxoplasmosis in The prevalence of toxoplasmosis in goats varies older age groups may be due to longer terms of exposure among the countries of the EU, but the data in Slovakia to infection, higher number of infection sources, greater appear rather sporadically. The data on the preva- possibilities of acquiring infection and others (Teshale lence of toxoplasmosis in goats within the EU (gathered et al. 2007; Carneiro et al. 2009) that is in concordance throughout 1990–1999) were compiled by Tenter et al. with our results. (2000). They reported the prevalence ranging from 19% Several authors (e.g., Jittapalapong et al. 2005; to 77%. The percentage of prevalence in the countries Teshale et al. 2007) observed a higher prevalence of depends mainly on eating habits of the relevant popu- toxoplasmosis in females comparing to males. Unlike lation. Wilson & McAuley (1999) reported that a high Bisson et al. (2000) and Cavalcante et al. (2008), who prevalence of toxoplasmosis can be associated with con- assume that gender does not present a significant risk sumption of undercooked meat. factor in T. gondii infection. Cavalcante et al. (2008) Recent studies carried out in Slovakia reported the and Carneiro et al. (2009) found that the main risk prevalence of toxoplasmosis in animals varying between factors in goats include age, breed, housing practices, 38% and 58.5% (Pleva et al. 2005; Ministry of Agricul- presence of cats and their access to feed. According ture of the Slovak Republic, 2009). 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