Molecular detection of Toxoplasma gondii infection in slaughtered ruminants (sheep, goats and cattle) in Northwest Tunisia

Accepted Manuscript Molecular detection of Toxoplasma gondii infection in slaughtered ruminants (sheep, goats and cattle) in Northwest Tunisia

Yosra Amdouni, Mohamed Ridha Rjeibi, Mariem Rouatbi, Safa Amairia, Sofia Awadi, Mohamed Gharbi PII: DOI: Reference:

S0309-1740(16)30383-7 doi: 10.1016/j.meatsci.2017.07.004 MESC 7309

To appear in:

Meat Science

Received date: Revised date: Accepted date:

12 October 2016 21 June 2017 2 July 2017

Please cite this article as: Yosra Amdouni, Mohamed Ridha Rjeibi, Mariem Rouatbi, Safa Amairia, Sofia Awadi, Mohamed Gharbi , Molecular detection of Toxoplasma gondii infection in slaughtered ruminants (sheep, goats and cattle) in Northwest Tunisia, Meat Science (2017), doi: 10.1016/j.meatsci.2017.07.004

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ACCEPTED MANUSCRIPT Molecular detection of Toxoplasma gondii infection in slaughtered ruminants (sheep, goats and cattle) in Northwest Tunisia

YosraAmdouni1*, Mohamed Ridha Rjeibi1, Mariem Rouatbi1, Safa Amairia1, Sofia Awadi2,

Laboratoire de Parasitologie, Univ. Manouba, Institution de la Recherche et de

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Mohamed Gharbi1

Thabet, 2020 Sidi Thabet, Tunisia. Regional Slaughterhouse of Béja, 9000, Tunisia.

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l’Enseignement Supérieur Agricoles, Ecole Nationale de Médecine Vétérinaire de Sidi

* Corresponding author

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Tel.: +216 71 552 200 (ext. 264); Fax: +216 71 552 441

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E-mail address: [email protected] (Y. Amdouni)

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ACCEPTED MANUSCRIPT Abstract The present study aimed to estimate the molecular prevalence of T. gondii infection in meat from slaughtered sheep, goats and cattle in Northwest Tunisia (Béja district). PCRs were performed on genomic DNA extracted from 420 meat samples (150 ewes, 120 goats and 150 cows). The overall molecular prevalence of T. gondii in sheep, goats and cattle were 33.3

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(50/150), 32.5 (39/120) and 19.3% (29/150), respectively. Toxoplasma gondii molecular

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prevalences in the three meat ruminant species were significantly higher in adults compared to

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young animals (p<0.001). The infection prevalence differed significantly within localities in sheep (p<0.001), goats (p<0.001) and cattle (p=0.019). These results provide important

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information about exposure of humans to T. gondii through the consumption of raw or undercooked ruminants’ meat. Extension programmes should be implemented to decrease the

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risk of infection related to sheep, goats and cattle meat manipulation and raw or undercooked

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meat consumption.

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Keywords

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Toxoplasma gondii, Sheep, Goats, Cattle, Meat, Tunisia.

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ACCEPTED MANUSCRIPT 1. Introduction Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii, a protozoan parasite belonging to the order Coccidia and the Apicomplexa phylum. Toxoplasma gondii is one of the most prevalent apicomplexan parasite of human and warm blooded animal species which infects up to one-third of the human population (Saadatnia & Golkar, 2012).

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The parasite represents a serious threat for seronegative pregnant women and female

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ruminants, it can cause congenital infections and abortion in both humans and livestock (Baril

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et al., 1999). T. gondii infection are widely prevalent in human and animals worldwide (Dubey & Beattie, 1988), it was rarely isolated from naturally infected cattle tissues (Esteban-

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redondo, Maley, Thomson, Nicoll, & Wright, 1999; Dubey et al., 2005; Dubey, 2010). Many studies were carried out in Tunisia to provide information about human exposure to T.

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gondii. In North Tunisia, where the climate is mild, the overall prevalence was estimated to 58.4% (Bouratbine et al., 2001). There was a significant correlation between the age and the

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seroprevalence; between 10 and 20 years, it increases from 24.5 to 52.1% respectively

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(Bouratbine et al., 2001). In Tunis district, almost half (47.7%, N= 2351) of the tested pregnant women were seroprevalence (Fakhfakh et al., 2013). In the district of Sousse

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(Central Tunisia), the seroprevalence in female high school students (mean age: 17.8 years) was 77% (Jemni et al., 1985). Between 1994 and 2006, the seroprevalence in Sfax district

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(South Tunisia) was estimated to 39.3% in 40,566 pregnant women (Sellami et al., 2010). Among them, 16 congenital toxoplasmosis cases were detected. Human infection is principally acquired by ingestion of food or water contaminated by oocysts or by consuming raw or undercooked meat containing tissue cysts (Montoya & Liesenfeld, 2004). Usually, the prevalence of T. gondii infection is higher in sheep than goats and cattle (Dubey & Streitel., 1976; Sharif et al., 2007).

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ACCEPTED MANUSCRIPT In Tunisia, culinary habits represent a risk factor of human’s toxoplasmosis. Indeed, beef, goats and lamb meat barbecues are very appreciated by Tunisian consumers; this cooking method does not inactivate T. gondii bradyzoites. For example, Khayeche, Mhadhbi, Gharbi, Nasfi, & Darghouth (2014) showed that in 10% of the studied families, at least one elderly person consumed raw fatty tail or liver and 2% of them consumed undercooked meat. Zhou &

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Tao. (2015) showed that barbecue consumption represent an important risk factor of T. gondii

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infection.

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To assess the infection risk by Toxoplasma gondii in Tunisian human population, we carried out the present study in order to estimate the molecular prevalence of T. gondii in meat

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samples of slaughtered ruminants in Northwest Tunisia (Béja district).

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2. Materials and methods

2.1. Study area and sample collection

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The present study was carried out in Béja district (Northwest Tunisia, latitude 36°43´N;

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longitude 9°10´E). This district covers an area of 3,740 km2; it has a humid climate with a mean annual rainfall of 626 mm. The mean minimum and maximum temperatures are 10 and

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23°C in January and August, respectively (National Institute of Meteorology, Tunisia). A total number of 420 meat samples were collected from neck muscles of 150 ewes, 120

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goats and 150 cows that were slaughtered in the regional slaughterhouse of Béja. The samples were placed in sterile, identified vials and stored at -20°C until analysed.

2.2. DNA extraction and polymerase chain reactions DNA was extracted from 50 mg of each meat sample using a commercial DNA extraction kit (Wizard® Genomic DNA Promega, Madison, Wisconsin, USA) according to the manufacturer’s instructions, then stored at -20°C until used. In order to confirm that DNA had

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ACCEPTED MANUSCRIPT been successfully extracted from meat samples, universal PCR was performed for each sample to detect the presence of any eukaryotic DNA (Wang, Tian, Gao, Bougouffa, & Qian, 2014). PCR reactions were performed with a mix consisting of 1x PCR buffer, 2 mM MgCl2, 10 µM of each primer (1A and 564R), 0.2 mM of each dNTP, 2 U Taq Polymerase (Vivantis, Chino, California), 1.5 μL of DNA template and distilled water to a total volume of 25 μL

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(Table 1). PCRs were performed in a thermocycler (Applied Biosystem 2700) at the following

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conditions: initial denaturation at 94°C for 5 minutes, followed by 25 cycles (94; 59 and 72°C

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for 50 seconds each) and a final extension of 72°C for 10 minutes.

Toxoplasma gondii specific PCR was performed using the protocol described by Boughattas

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et al. (2014) with a set of primers that amplifies a 114 bp B1 gene region (Table 1). PCR reactions were carried out in 25 µL of mixture consisting of 1x PCR buffer, 2.5 mM MgCl2,

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200 µM dNTP each, 0.6 mg BSA, 10 pmol of primers, 1 U of Taq polymerase and 5 µL of sample DNA.

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Amplification was carried out during 2 min at 50°C then 6 min at 95°C followed by 40 cycles

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(denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec and an extension step at 72°C for 1 min adding 1 sec/cycle) with a final extension at 72°C for 7 min. Positive (Toxoplasma

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gondii DNA) and negative (distilled sterile water) controls were included in each PCR run. The amplicons were detected by gel electrophoresis using 3% (w/v) agarose then visualized

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under ultraviolet light.

2.3. Statistical analysis The infection prevalences were compared using the chi-square Mantel-Haenszel test, with Epi Info 6 software at 5% threshold (Schwartz, 1993). Risk factors were evaluated using stratified odds ratio (Ancelle, 2006).

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ACCEPTED MANUSCRIPT 3. Results The overall T. gondii infection prevalence rate in sheep, goats and cattle were 33.3; 32.5 and 19.3%, respectively (Table 2) (p=0.011). The infection rate was significantly higher in sheep aged of more than one year (49.3±11.3%) than the other age groups (p<0.001). The highest prevalence was observed in Téboursouk and Thibar localities (53.2±14.3%) (p<0.001) (Fig.1).

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Barbarine was the most infected sheep breed (68.9±13.5%) (p<0.001).

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The highest and lowest goats’ meat infection prevalence was observed in South Béja

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(51.7±12.6%) and Amdoun (26.7±15.8%) localities (p<0.001) (Fig.1). The infection rate was significantly higher in goats aged of less than three years (66±17.5%) than other age groups

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(p<0.001). Infection prevalence in exotic breed goats was higher (55±21.8%) than local breed goats (16.7±13.3%) (p=0.017).

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In cattle, the highest prevalence (33.3±16.9%) was observed in cattle aged of more than eight years. Whilst, the lowest prevalence (3.7±5%) was observed in cattle aged between two and

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eight years (p<0.001). Cattle from South Béja were the most infected animals (28.6±13.7%)

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(p=0.019) (Fig.1).

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4. Discussion

Several molecular studies were carried out to estimate the molecular prevalence of T. gondii

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in Tunisian sheep. Gharbi et al., (2013) estimated the molecular prevalence in two Tunisian regional slaughterhouses to 12.7 and 25.5% in Ben Arous and Sidi-Bouzid, respectively. Khayeche et al. (2014) showed that the molecular prevalence of T. gondii in lambs slaughtered by 70 families in Central-East of Tunisia during the religious Muslim feast was low (5.7%). This result was explained by the low age of the slaughtered animals. Indeed, 92.9% of animals were aged of less than one year. In Tunis City, T. gondii molecular prevalence in sheep was higher (50%) than our findings (33.3±7.5%). This discrepancy may

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ACCEPTED MANUSCRIPT be explained by the highest density of cats in Tunis City (Boughattas et al., 2014). Similar infection prevalence was reported in pregnant women in North Tunisia, Tunis City, Sousse (Centre Tunisia) and Sfax (South Tunisia) (58.4; 47.7; 77; 39.3 and %, respectively) (Jemni et al., 1985; Bouratbine et al., 2001; Sellami et al., 2010; Fakhfakh et al., 2013). Berger et al. (2007) argued that T. gondii seroprevalence among pregnant women in the same area was

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correlated by the seroprevalence in sheep.

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Lower prevalence was reported in Portugal, Spain, Ireland and Switzerland (17.6; 5.4; 3.6 and

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3.3%, respectively) (Berger-schoch et al., 2011; Moreno et al., 2012; Halová et al., 2013; Lopes et al., 2015 ). These lower infection rates could be explained by the presence of low

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exposure to the infective oocysts in the environment (Andrade, Carneiro, Medeiros, Neto, & Vitor, 2013; Hammond-Aryee, Van Helden, & Van Helden, 2015).

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Significant association was observed between the animals’ ages and T. gondii infection prevalence. As reported by Asgari & Sarnevesht (2011) and Boughattas et al. (2014), we

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found an age-prevalence positive correlation arguing that sheep mostly acquired the infection

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progressively after birth (Halos et al., 2010). For goats and cattle, no molecular studies were carried out in Tunisia to estimate the

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prevalence of T. gondii infection despite the high quantities of goats’ and beef produced meat (9,600 and 58,000 Tonnes/year, respectively) (FAO, 2016).

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The prevalence in Tunisian goats (32.5±8.3%) was higher than those reported in Southern Iran (22.5%) and Brazil (13.7%) (Silva et al., 2009; Asgari & Sarnevesht, 2011), and lower to the estimation of Lopes et al. (2015) in Northern Portugal (60%). This might be attributed to the Portuguese climate, which is characterized by a higher rainfall that increases oocysts’ persistence and dispersion. This high T. gondii prevalence in goats raises the possibility of infection transmission through the manipulation of raw meat or the consumption of goats’ undercooked meat.

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ACCEPTED MANUSCRIPT In goats, the highest and lowest T. gondii infection prevalence were observed in exotic (55%) and local breed (16.7%), respectively (p=0.017). These results may be due to a higher genetic resistance of local breeds to T. gondii infection in comparison with exotic breeds. Significant association was observed between the animals’ ages and T. gondii infection prevalence. The infection prevalence was higher in goats aged between four and five years (33.3%; p<0.001)

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than the other age groups.

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In cattle, the prevalence of T. gondii (19.3±6.3%) was by far higher to those reported in Brazil

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and Switzerland (2 and 3.8%, respectively) (Santos, Costa, & Gondim, 2010; Berger-schoch et al., 2011), and lower than values reported in Colombia and Northern Portugal (36.6 and

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50%, respectively) (Lopes et al., 2015; Franco-Hernandez, Acosta, Cortés-Vecino, & GómezMarín, 2016). The infection prevalence was higher in cattle aged of more than eight years

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(33.3±16.9) (p<0.001) than the other age groups. These results may be explained by the fact that animals clear the infection with age and become resistant to new infections.

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Dubey et al. (2014) affirmed that humans become infected by ingesting tissue cysts from

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undercooked meat or by consumption of food contaminated with sporulated oocysts. A systematic review was carried out in the context of the Eurotoxo project to evaluate the risk

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factors associated with Toxoplasma gondii contamination in pregnant women showed that consumption of undercooked meat was considerate as an important risk factor of human

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contamination (Leroy & Hadjichristodoulou, 2005). In France, in the same context, a study carried out by Baril et al. (1999) on 180 pregnant women showed that consumption of undercooked meat of sheep and beef is an important risk factor. In Tunisia, Fakhfakh et al. (2013) showed that 47.7% of the seropositive persons were consuming undercooked meat. Due to the scarcity of data concerning T. gondii infection in Sheep, cattle and goats, our results are important to improve the risk assessment of human contamination through sheep, beef and goats’ meat manipulation and consumption. Many studies showed that T. gondii

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ACCEPTED MANUSCRIPT DNA can be detected in meat cattle (Aspinall, Marlee, Hyde, & Sims, 2002; Canada et al., 2002; Moré, Basso, Bacigalupe, Venturini, & Venturini, 2008). In the present study, the regional prevalence differed significantly in sheep, goats and cattle (p<0.001).

5. Conclusion

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Our results provided an estimation of molecular T. gondii prevalence in sheep, goats and

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cattle Tunisian meat. Based on the high molecular prevalence in Tunisia, T. gondii meat cysts from these three species must be considered as an important source of human infection and

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added to the list of pathogens transmitted by raw or undercooked ruminants’ meat.

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Educational programmes must be established to inform Tunisian human populations about the risk of manipulating or consuming raw and undercooked meat (mainly barbecue), not only of

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sheep, but also of goats and cattle. Medical doctors, veterinarians and human Tunisian

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Acknowledgment

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population should be informed about the high infection levels in these three species.

The study was funded by “Epidemiology of endemic infection of herbivores in Tunisia:

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application to the control”. Ministry of Higher Education, Scientific Research and Information, Tunisia. The authors thank Mr Limam Sassi, Mr Bechir Guesmi and Mr Taoufik

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Lahmar for their technical support.

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ACCEPTED MANUSCRIPT Zhou, J., & Tao, L. (2015). Seroprevalence and risk factors of Toxoplasma gondii infection among pregnant women in Wuxi region. Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi

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= Chinese Journal of Schistosomiasis Control, 27(6), 604–7.

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ACCEPTED MANUSCRIPT Figure caption Fig.1. Sheep, goats and cattle PCR infection prevalence of Toxoplasma gondii in the Béja

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locality (North-West Tunisia).

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ACCEPTED MANUSCRIPT Table 1: Primers used for detection by PCR of universal and Toxoplasma gondii from sheep, goats and cattle in this study. Target gene 18S

Primer name 1A 564R B22

AACCTGGTTGATCCTGCCAGT

Reverse primer

TGGGTCTACGTCGATGGCATGACA AC

Reference

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Boughattas et al. (2014)

Wang et al. (2014)

GGCACCAGACTTGCCCTC AACGGGCGAGTAGCACCTGAGGAG A

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B23

Forward primer Reverse primer Forward primer

Product size (bp) 600

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B1

Primers 5’—3’

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Toxoplasma gondii

Type

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Primer Specificity Universal PCR

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ACCEPTED MANUSCRIPT Table 2: Association between toxoplasmosis prevalence in sheep, goats and cattle in Béja (North-West Tunisia) and different parameters based on PCR.

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<0.001*

0.29 [0.09 ; 0.60]

<0.001*

NA

<0.001*

0.08 [0.01 ; 0.33]

<0.001*

0.29 [0.10 ; 0.69]

0.017*

NA

0.019*

0.10 [0.03 ; 0.30]

0.0009*

0.34 [0.1 ; 0.94]

0.074

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Cattle

14/76 (18.4±8.7) 31/45 (68.9±13.5) 5/29 (17.2±13.7) 0/30 (0) 8/30 (26.7±15.8) 31/60 (51.7±12.6) 18/30 (66±17.5) 20/60 (33.3±11.9) 1/30 (3.3±6.4) 5/30 (16.7±13.3) 23/70 (32.9±11.0) 11/20 (55±21.8) 13/54 (24.1±11.4) 4/27 (14.81±13.4) 0/27 (0) 12/42 (28.6±13.7) 2/54 (3.7±5.03) 17/66 (25.7±10.5) 10/30 (33.3±16.9) 12/39 (30.8±14.5) 11/77 (14.3±7.8) 2/21 (9.5±12.5) 4/13 (30.8±25.1)

0.22 [0.09 ; 0.48]

<0.001*

AC

Goats

Barbarine Noire de Thibar Cross breed Locality Amdoun Téboursouk South Béja Age group ≤3 (years) 4 to 5 ≥6 Breed Local breed Cross bred Exotic breed Locality Amdoun Téboursouk and Thibar North Béja South Béja Age group <2 (years) [2-8[ ≥8 Breed Cross breed Holstein Brown Swiss Tarentaise SEa: Standard Error ORb: Odds Ratio 95% C.Ic.: 95% Confidence Interval NAd: not applicable * Statistically significant (p ≤ 0.05)

D

Breed

P value

PT

Age group (years)

Amdoun Téboursouk and Thibar North Béja South Béja <1 ≥1

Stratified OR b [95% CI]c NAd

RI

Locality

SC

Sheep

Positive/examined (%±SEa) 18/45 (40±14.3) 25/47 (53.2±14.3) 0/20 (0) 7/38 (18.4±12.3) 13/75 (17.3±8.6) 37/75 (49.3±11.3)

NU

Parameter

MA

Species

17

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

Fig 1.

18

ACCEPTED MANUSCRIPT Highlights The molecular prevalence of Toxoplasma gondii in slaughtered sheep, goats and cattle collected from North-West Tunisia was estimated. The molecular prevalence in sheep, goats and cattle were 33.3 (50/150), 32.5 (39/120) and 19.3% (29/150), respectively

PT

Toxoplasma gondii meat cysts must be considered as an important source of infection in

AC

CE

PT E

D

MA

NU

SC

RI

humans

19