Molecular characterization of Toxoplasma gondii and Sarcocystis spp. in raw kibbeh and other meat samples commercialized in Botucatu, Southeastern Brazil

Toxoplasmosis occurs worldwide causing economic losses to the animal production and problems to the public health. The study aimed to detect Toxoplasma gondii and Sarcocystis spp.in 141 meat products from commercial meat cuts of pork, beef, and kibbeh sold in commercial markets from Botucatu, SP, Brazil. Samples were bioassayed in mice to isolate the parasite, and the parasite DNA detected by PCR targeting the 529 base pairs repeat element region (PCR-529-bp). All samples resulted negative on bioassay, whereas PCR positive for 9 (6,38%), distributed as 5/48 beef, 3/49 pork, and 1/44 kibbeh. PCR-positive were investigated for the the parasite genotype using multiplex-, nested-, and RFLP-PCR for 11 markers (SAG1, 5’-3’SAG2, alt.SAG2, SAG3, B-TUB, GRA6, L358, c22-8, c29-6, PK1, Apico). Complete genotype was determined on just one PCR-positive sam le that matched MAS, TgCkBr89 and TgCkBr147 isolates already identified. In addition, nested- and RFLP-PCR targeting 18S rRNA was run for all PCR-positive samples and, the products, sequenced and aligned to the GenBank at NCBI website. Four samples showed 100% homology with T. gondii (GenBank #L37415.1), three with Sarcocystis hominis (GenBank #AF006471.1), two Sarcocystis cruzi (GenBank #AF176934.1), and one Sarcocystis hirsuta (GenBank #AF006469.1), indicating the circulation of T. gondii and Sarcocystis spp.


Introduction
Toxoplasma gondii is an obligate intracellular protozoan parasite, which causes toxoplasmosis, a worldwide distributed zoonosis, infecting homoeothermic hosts, including humans (Acha & Szyfres, 2003). Felids are the definitive hosts, while livestock animals develop tissue cysts (bradyzoites) in different tissues, e.g. the musculature (intermediate hosts). Contaminated food increases the importance of this infection to the public health. This anthropozoonosis can cause severe disease on newborns and immunosuppressed patients (Millar et al., 2007). Many different risk factors can be associated to this infection, including the ingestion of food containing the cysts as well as its handling by butchers and people who manipulate it.
Sarcocystis spp. are obligate protozoan parasites causing sarcocystosis, another worldwide distributed zoonosis. More than hundred species of Sarcocystis infect mammals, but only two are known to parasitize the human intestine, ie. S. suihominis and S. hominis. Other species, ie. S. hirsuta, S. miescheriana, S. gigantea, S. cruzi, and others, affect animals (Acha & Szyfres, 2003). All Sarcocystis spp. have obligatory heteroxenous life cycle. Carnivores and omnivores are usually definitive (sporogony -sexual stage) hosts while omnivores and herbivores are intermediate hosts (merogony -asexual stage). The definitive host acquires them upon ingesting meat from intermediate hosts infected with mature cysts (sarcocysts), containing the parasite (infective bradyzoites). The intermediate host acquires the infection upon consuming oocysts or mature sporocysts shed by the definitive hosts in their feces (Lindsay et al., 1995;Dehkordi et al., 2017).
It is extremely important to search for T. gondii (Acha & Szyfres, 2003) and Sarcocystis spp. (Dehkordi et al., 2017) in meat products used for human consumption since they constitute a transmission route responsible for foodborne outbreaks (Acha & Szyfres, 2003). The infection in livestock animals can be detected by serological tests and bioassay in mice (Kijlstra & Jongert, 2009). Serology may be used to have a screening of the T. gondii infection (Acha & Szyfres, 2003). de Macedo et al. (2012) observed 48.3% dairy cows and 3.7% fetuses seropositive for T. gondii using the indirect fluorescent antibody technique (IFAT), while bioassay in mice resulted positive in only 10% dairy cows and 23.3% fetuses. Spagnol et al. (2009) reported 11.83% seropositive cattle among 600 slaughtered animals compared to 41.4% observed by Daguer et al. (2004)  Since meat products participate to the transmission chain of toxoplasmosis to animals and humans, the present study aimed to detect T. gondii and Sarcocystis spp. in meat products sold at commercial markets in Botucatu, SP, Brazil.

Sampling
Meat samples were sampled from different markets from Botucatu, a city placed at the Mid-West region of Sao Paulo State, at the Southeastern Region from Brazil (22º52'20"S; 48º26'37"W). Samples of pork, beef cuts, and raw kibbeh were obtained from four butcher shops (A, B, C and D) and one supermarket, monthly, along 10 months to increase the diversity of the origin of the commercialized meat products sold by the suppliers. One hundred forty-one meat cuts were sampled, as follows: swine gammon (49), bovine shank (48), and raw kibbeh (44). Samples were individually packed in sterile plastic bags, identified, and stored in an isothermal box, at 4°C, and processed within 1 hour. The present study was approved by the Ethics Committee on Animals Use (CEUA) of the School of Veterinary Medicine and Animal Science, UNESP, Botucatu, SP (#155/2010).

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Toxoplasma gondii and Sarcocystis spp. in raw meat Bioassay in mice All meat samples were bioassayed in 30 day-old, female albino Swiss mice (Mus musculus). The samples were weighed (50 g) and eight fragments from different sites of each meat sample were pooled and mixed. Each pool (10 g) was individually ground, diluted 1:5 (w/v) in 0.18% saline solution and digested by the acid action of pepsin (Dubey, 1998). Aliquot of each pool (1 mL) was stored at -80°C for molecular assay. Five mice per pool were bioassayed with 1 mL suspension (per mice), using subcutaneous inoculation. Bioassayed mice were observed along 60 days. After the observational period, blood samples were drawn from all alive mice by the retro-orbital sinus puncture into a microtube without anticoagulant. The blood samples were centrifuged at 1600 x g for 10 minutes, and the sera sample searched for T. gondii IgG antibodies using a serological test with a cut-off 1:16. All died mice, or mice inoculated with meat cut positive for T. gondii DNA detection, had the brain removed and also searched of T. gondii DNA using molecular technique. Samples were considered positive after detected T. gondii DNA or T. gondii IgG antibodies.

Modified agglutination test (MAT)
T. gondii IgG antibodies were searched in the blood serum samples of bioassayed mice using modified agglutination test (MAT) (Desmonts & Remington, 1980), on 96-well U-shaped-bottom microplates (Cralplast, Brazil). The cut-off titer was 16. Positive result was considered after the formation of a pellicle covering at least half of the well, and negative when a sediment ("button" or a "ring") was formed at the bottom of the well.

Detection of T. gondii using PCR of the 529 bp fragment (PCR-529-bp)
DNA extraction and purification of the meat samples were done using the extraction kit Illustra Tissue and cells genomic Prep Mini spin kit (GE Healthcare, USA), according to the manufacturer instructions. The concentration and the quality of the extracted DNA were determined using a Nanovue spectrophotometer (GE Healthcare, USA).
PCR-529-bp was assayed using TOX4 and TOX5 primers, targeting 529-base pairs (bp) repeat element region, which is a 200-to 300-fold repetitive in the parasite genome, and result on an amplification product of 529-bp (Homan et al., 2000). All amplified products were visualized on electrophoresis using 1.5 agarose gel. The products were visualized using the digital documentation system, Gel-Doc-it System (UVP, USA), and VisionWorks Software (UVP, USA).

Sequencing
The products of PCR for primers TOX4 and TOX5, and that of nested-PCR for gene 18S rRNA were purified using the purification kit Centrifugal Filter Units, MRCF0R030 (Millipore, USA) or ExoStar1-Step, US77705 (GE Healthcare, USA). DNA was quantified using the spectrophotometer Nanovue (GE Healthcare, USA), and electrophoresed on agarose gel.
Sequencing was run on an automated sequencer, Applied Biosystems 3100 (Applied Biosystems, USA), employing ABI BigDye kit and GE DYEnamic ET terminator kit (GE Healthcare, USA), and analyzed by the software Sequence 4/9 Toxoplasma gondii and Sarcocystis spp. in raw meat Analyzer, using Base Caller Cimarron 3.12. The obtained sense and antisense sequences were aligned and visualized using the software BioEdit Sequence Alignment Editor 7.0.9.0. Nucleotide sequences were analyzed using Nucleotide Basic Local Alignment Search Tool (BLASTn) and compared to those deposited on GenBank nucleotide database.

Statistical analyses
All data were tabulated on Excel spreadsheet. Fisher's exact test was used to analyze the association of the PCR results, the type of meat sample (beef, pork and kibbeh), and the origin of samples (butcher shops or supermarket). A significance level of 5% was adopted (Triola, 2005).

Discussion
The search for zoonotic agents in meat for human consumption has high importance, considering its risk to public health (Acha & Szyfres, 2003). In natura meat products, and those derived from beef and pork, are important protein sources for humans. Livestock animals play an important role to the spread of T. gondii infection, since they harbor tissue cysts containing bradyzoites and constitute one of the possible transmission routes to humans. The ingestion of viable tissue cysts by a non-immune host may result in an infection with the parasite (Tenter, 2009).
Tissue cyst formation varies among animals and is related to the parasite organotropism (Dubey & Jones, 2008). The cysts present in carcasses and meat products remain viable and infective for a variable time. However, bioassay in mice requires a minimum infective dose, the virulence, and the absence of agents that inactivate the parasite to isolate the parasite (Mendonça et al., 2004).  Toxoplasma gondii and Sarcocystis spp. in raw meat The PCR-positive and bioassay-negative results in the present study indicate presence of the parasite DNA on the inoculated material, but its viability was compromised. The same was observed by Mendonça et al. (2004). These authors detected T. gondii DNA in 70 sausage samples sold in commercial markets, but all samples resulted negative to the bioassay in mice. Thus, the study on commercialized meat products must take into consideration the conservation period and status of the investigated meat. Although it is known that freezing inactivates bradyzoites. According to Bayarri et al. (2012), freezing is enough to inactivate T. gondii tachyzoites and bradyzoites.
The search for T. gondii in meat is important to evaluate the transmission chain of this protozoan, especially on raw kibbeh. Raw kibbeh is usually consumed in natura. This increases the risk to the consumers, once this type of meat is prepared from different species, i.e., sheep . Benitez et al. (2017) reported higher seroprevalence for T. gondii antibodies in dog owners (41.54%) from Londrina, PR, Brazil, than their dogs (16.32%) (p<0.01). Concerning some analyzed variables, 97.7% owners reported meat consumption and occasional undercooked meat in common dishes, ie barbecue (32.8%), fried meat (24.5%), and raw kibbeh (17,8%), while just 30.2% offered sporadically meat to the dogs. This important data reflects the importance of the food care after buying meat cuts or products even on butcher shops or supermarkets. The identification of T. gondii and Sarcocystis spp. in beef and sheep, as kibbeh, products does alert the population to adopt control measures and meat treatments before consuming or offering to the pets.
The consumption of kibbeh may be important to the occurrence of several food-borne outbreaks involving meat products, including toxoplasmosis (Bonametti et al., 1997;Cook et al., 2000;Kijlstra & Jongert, 2008). On a public health concern, Hashimoto Pugliesi et al. (2020) observed 16.9% pregnant women from Maringa, PR, eat raw or undercooked meat, 11.8% eat raw kibbeh, and 27.2% eat rare barbecue. Even with low rates, the T, gondii susceptibility is there. Bonametti et al. (1997) reported an outbreak with 17 human Toxoplasma cases after ingestion of raw kibbeh on a party in Bandeirantes, PR, Brazil. All patients presented T. gondii IgM and IgG antibodies.
T. gondii infection, with no co-infection, was detected in pork and kibbeh samples. Pigs and sheep are some of the most important animals related to the toxoplasmosis transmission chain, involving meat products (Tenter et al., 2000). In contrast to Brazil, livestock animal from Colombia are high exposed to the parasite. Franco-Hernandez et al.
Concerning the meat products from cattle, T. gondii (PCR-529-bp) and Sarcocystis spp. (sequencing, 18S rRNA) were also detected in this study. Three distinct Sarcocystis species were identified, including S. cruzi, S. hirsuta and S. hominis. These results show the relevance of bovine meat to the public health, since T. gondii and S. hominis have a zoonotic impact. Cattle are intermediate hosts of Sarcocystis sp. (Carlton & McGavin, 2005), being affected all three identified species, e.g., S. cruzi, S. hominis and S. hirsuta (Lindsay et al., 1995). The macroscopic observation of the parasite on bovine musculature is crucial to diagnose the parasitosis during meat inspection at the slaughterhouse (Fortes, 2004).
Genotyping by employing 11 genotypic markers has been adopted worldwide for T. gondii DNA detection and analysis. Reports from South America have indicated significant genotypic variability identified by this technique, which allowed the characterization and phylogeny of different T. gondii isolates (Da Silva et al., 2009). Several studies have confirmed the high genetic diversity of the parasite in South America since it has been isolated from several animal species, such as goats from Ceará, Brazil (Cavalcante et al., 2007), dogs and cats from Colombia (Dubey et al., 2006a, Dubey et al., 2007a, and chicken from Nicarágua (Dubey et al., 2006b) and from Brazil (Dubey et al., 2007b). In Mid-West region of Sao Paulo State, Brazil, T. gondii isolates from sausages have already been characterized using molecular techniques (Vieira Da Silva et al., 2005).
According to the genotyping, T. gondii isolate was characterized as atypical and matched to MAS, TgCkBr89 and TgCkBr147, already identified isolates. MAS was isolated from a human patient in France, and it was classified as virulent (Grigg et al., 2001). In addition, TgCkBr89 was isolated from chickens from Rio de Janeiro State, Brazil (Dubey et al., 2003), whereas TgCkBr147 was isolated from chickens from Rio Grande do Sul, Brazil (Dubey et al., 2007b), both isolates virulent to mice and genotyped by . The other PCR-positive samples did not present RFLP-PCR patterns to determine the T. gondii genotype. This information suggests a low quantity of T. gondii DNA. Studies researching new genotypes in production animals must be encouraged in order to understand the dynamics of the distribution of classic and atypical T. gondii genotypes, contributing to the active vigilance and epidemiology of toxoplasmosis, especially using molecular epidemiology.
For the prophylaxis of toxoplasmosis, other measures are evidently related to the epidemiological transmission chain of this zoonosis, i.e., the care of definitive hosts like primoinfected felids (domestic cat). Domestic cat is