Prevalence of Toxoplasma gondii Antibodies in Individuals Occupationally Exposed to Livestock in Portugal

Toxoplasmosis is a worldwide zoonotic disease with different and complex routes for transmission. Workers occupationally exposed to animals or raw meat and viscera (WOE) may be at more risk than the general population, however conflicting data exist on the risk of developing toxoplasmosis due to this close contact. To add knowledge to this topic, the aim of the present study was to ascertain if WOE were more likely to be anti-T. gondii IgG seropositive than the general population as well as to study risk factors for T. gondii infection such as professional activity, gender, age, years of work and region. For this purpose, a case–control study using archived samples was setup. A total of 114 WOE (including pig slaughterhouse workers, butchers, veterinarians and farmers) and 228 anonymous volunteers (matched with cases by region, age and gender) were studied for anti-T. gondii IgG. A significantly higher anti-T. gondii IgG occurrence (p = 0.0282) was found in WOE when compared with the general population (72.8% [CI = 64.6–81.0%] versus 60.1% [CI = 54.6–65.6%]). Multivariate analysis showed that WOE of more than 50 years of age were more likely to be seropositive for anti-T. gondii IgG (aOR = 16.8; 95% CI 3.6–77.5; p < 0.001) than those aged less than 50 years. To our knowledge, this is the first case–control study on the prevalence of anti-T. gondii IgG in WOE in Portugal, also showing an added risk for T. gondii infection in those exposed to animals or their meat and viscera.


Introduction
Toxoplasmosis is considered to be one of the most important parasitic zoonoses worldwide [1]. Toxoplasma gondii is a facultative heteroxenous protozoan with a complex life cycle and multiple transmission routes, capable of infecting, hypothetically, all warmblooded animals, including humans [2,3]. Although infection by T. gondii is typically asymptomatic, it might cause life-threatening disease in the immunosuppressed, and abortion/congenital disease of the foetus in pregnant women infected by the parasite for the first time during pregnancy [4]. These facts justify the need to determine T. gondii occurrence not only in humans but also in animals that act as reservoirs. In domestic animals, congenital toxoplasmosis affects especially small ruminants (essentially sheep) and pigs, causing abortion and congenital malformation of foetuses [4][5][6].
In the last few years, determining risk factors for T. gondii infection in humans has been a priority for investigators. This allowed identification of contact with soil, age, poor hygiene, consumption of raw and undercooked meat, drinking of untreated water, consumption of uncleaned vegetables and contact with animals [1,[7][8][9] as some of the most important risk factors. A few epidemiological serosurveys tried to ascertain the risk for T. gondii infection in individuals in close contact with animals and their raw meat and viscera [10,11]. Despite this, the majority of these surveys are descriptive and only a few case-control studies have been developed [10,12]. Nevertheless, different professional activities have been suggested to be particularly at risk of T. gondii infection. For example, slaughterhouse workers, who can inadvertently ingest raw meat and be contaminated via trophozoites through wounds, when no protection materials are used, or contact with sporulated oocysts in animal fur during flaying, and veterinarians, because of contact with cats, as well as small ruminant placenta, or performing necropsy without protection [2,8,9,[13][14][15][16][17]. As such, the risk of exposure is dependent on the type of animal, the hygiene of the workers and the function they perform (evisceration, flaying, carcass cutting) [10,16,18,19].
To increase the knowledge of this important zoonosis, we aimed to ascertain if workers occupationally exposed to animals or raw meat and viscera (WOE) were more prone to T. gondii seropositivity than controls, by developing a case-control study on anti-T. gondii IgG in WOE and matched general-population controls.

Results
From the 114 WOE sera samples tested, 83 are positive for the presence of anti-T. gondii IgG antibodies. From the 228 control samples tested, 137 are positive for the presence of anti-T. gondii IgG antibodies ( Table 1). Seroprevalence of anti-T. gondii IgG in the WOE and control groups is 72.8% (CI = 64.6-81.0%) and 60.1% (CI = 54.6-65.6%), respectively. Chi-square test with Yates's correction was found to be 4.8181, showing that the difference in anti-T. gondii IgG seroprevalence between WOE and controls is statistically significant (p = 0.0282). The association between the detection of anti-T. gondii IgG in the group of WOE and the variables (region, gender, age group, profession and number of years of work) was evaluated by binomial logistic regression (univariate) and multinomial logistic regression analysis (multivariate) ( Table 2).
Being more than 50 years old (cOR 14.9; 95% CI 3.3-66.3; p < 0.001) was found to be associated with anti-T. gondii IgG seropositivity in binomial logistic regression (univariate analysis). Multivariate analysis also shows that the only risk factor for anti-T. gondii IgG seropositivity was the older age of the WOE (>50 years) with an aOR of 16.8 (95% CI 3.6-77.5; p < 0.001). None of the other variables concerning professional activity (slaughterhouse worker vs. butcher/veterinarian/farmer), gender (female vs. male), region (Centre vs. North) and years in practice (>16.5 vs. ≤16.5) are observed to be a significant risk factor for anti-T. gondii IgG seropositivity in WOE, by both univariate or multivariate analysis.

Discussion
In the present study, we report a significantly higher (p = 0.0282) anti-T. gondii IgG seroprevalence in WOE (75.8%; CI = 64.6-81.0%) compared with the general population (60.1%; CI = 54.6-65.6%). However, when ascertaining variables that could potentially be risk factors for higher seroprevalence of anti-T. gondii IgG, only age (>50 years old) was considered to be significant (p < 0.001).
The significant difference in anti-T. gondii IgG seroprevalence found in WOE and the general population (75.8% vs. 60.1%) was not surprising since other seroepidemiological surveys have reported similar results, with higher seroprevalences in those exposed to animals. A recent study from central India reported an anti-T. gondii seroprevalence of 48.9% for veterinarians and 48.4% for slaughterhouse workers against 6.6% in the control group [15]. This was also found to occur in Nigeria, where an anti-T. gondii IgG seroprevalence of 55.8% in WOE (slaughterhouse workers) vs. 32% of the general population was reported [16]. In Iran, butchers presented 48.8% anti-T. gondii IgG vs. 28.8% in the control group [12]. A systematic review performed in 2020 supports these findings, affirming that WOE had, in general, higher anti-T. gondii seroprevalence compared with general population. Despite this, evaluating risk factors frequently posed multiple difficulties mainly due to their complex relationship [7]. Although our results are consistent with various other studies, other authors have provided distinct conclusions. In a study from 2011, significant differences between WOE (slaughterhouse workers and butchers) and the control group were not found, showing no evidence that working with raw meat increased the probability of infection by T. gondii [10]. A few years later, the same team again presented data supporting lack of evidence for anti-T. gondii IgG association with WOE [13]. Other studies from 2008 and 2016 also reported no evidence for anti-T. gondii IgG differences between WOE and controls [19,20]. Of note, it was interesting to find a high anti-T. gondii seroprevalence in the controls of the present study (60.1%), which was higher than in the controls of other countries (6.6% to 32%) [12,15,16]. However, caution must be taken on this comparison as this might be the reflection of using distinctly aged groups or immunoassays with different sensitivities and specificities. A high seroprevalence in the Portuguese controls may be due to the important circulation of T. gondii in domestic animals and the consumption habits of the population [21].
When investigating risk factors for anti-T. gondii seropositivity in WOE, only age (>50 years old) shows a positive association, with an aOR of 16.8 (95% CI 3.6-77.5; p < 0.001). Hence, workers of more than 50 years of age are considered to have a 16.8-fold increased likelihood of having anti-T. gondii IgG than those less than 50 years old. Other studies reported age as a risk factor for the presence of anti-T. gondii IgG in WOE, namely in Japan, Malaysia, Iran, Finland and Poland, but all with a different cut-off age and focusing on different professions, from veterinarians to farmers [14,17,19,22,23]. This increased risk with age is not unexpected and is likely due to the longer period of exposure to risk factors, cumulatively occurring during the life-time exposure. Interestingly, years of practice did not appear to be a significant risk factor in the present study, as was in other reports [14].
In the present study, despite the inclusion of variables related to occupational exposure, several risk factors for the presence of anti-T. gondii IgG were not considered, such as those relating to consumption habits (raw vs. cooked meat, sheep/pig/chicken meat vs. beef) and hygiene at home (in the management of vegetables, water and cats), all of which are considered to be important [1].
To the best of our knowledge, this is the first epidemiological serosurvey searching for anti-T. gondii IgG in workers occupationally exposed to animals and their raw meat and viscera carried out in Portugal, showing that these WOE have an increased risk of infection with T. gondii compared with the general population controls. It is crucial that workers that are at continued risk of exposure re-enforce hygiene measures at work, such as not eating during labour time, and using protection equipment like gloves, masks and protective glasses [24]. Educating farmers about the existence of T. gondii and its importance, having a controlled and protected farm (strong biosecurity not allowing the presence of cats) and using clean water for crops are likely to have a positive influence in reducing the risk for T. gondii infection in animals and ultimately humans [1,4].

Sampling
For this study, archived (−80 • C) sera from a previous study were used [25]. WOE from the North and Centre of Portugal (n = 114) were selected, including 96 slaughterhouse workers (swine slaughterhouse), 5 butchers, 11 veterinarians and 2 farmers. Retrieved participants information included region (North or Centre of Portugal), gender (female or male), age (≤50 or >50 years old), professional activity (slaughterhouse worker or butcher/veterinarian/farmer) and number of years performing that job (≤16.5 or >16.5 years). The participant's age ranged from 20 to 83 years, and they had been working with animals for 1 month up to 55 years. For levels dividing the variable "years of work" (≤16.5 or >16.5 years) and age (≤50 or >50 years old), the median of each distribution was identified and used. Descriptive data regarding selected WOE can be found in Table 3. Of the control group, archived sera samples from anonymous volunteers (n = 228) matched with the WOE by region, gender and age were used in the proportion of two controls to each member of the WOE group. All procedures were conducted in accordance with the recommendations outlined in the Declaration of Helsinki and were approved by a national ethics board in Portugal (Comissão de Ética para a Saúde CHSJ; reference number: 99/2015).

Detection of Anti T. gondii Antibodies
All sera samples were individually tested for the presence of anti-T. gondii IgG antibodies using a commercial semiquantitative enzyme immunoassay (Toxoplasma ELISA IgG G1027, Edition 2018, Vircell, Granada, Spain). This test contains purified T. gondii antigen RH (ATCC 50174), and, according to the manufacturer it has a sensitivity of 98% (88-100%, confidence interval [CI] = 95%) and a specificity of 100% (89-100%, CI = 95%). All the procedures were performed according to the manufacturer's instructions.

Data Analysis
Data processing was firstly performed using Microsoft Office 365 Excel. After organizing all the data, the statistical analysis was performed using IBM SPSS version 28.0.0.0 statistical software. A CI was established at 95%. A chi-square with Yate's correction test for homogeneity of proportions was used to calculate significant differences in anti-T. gondii IgG seroprevalence between the WOE group and the control group. Binary and multinomial logistic regression analyses were carried out to determine which of the variables (region, gender, age, profession and years of work) were significantly (p < 0.05) associated with the detection of anti-T. gondii IgG among the WOE. Funding: This study was supported by FCT-Foundation for Science and Technology, I.P., through research grant PTDC/CVT-CVT/0228/2020 and by funds from the Global Health and Tropical Medicine (GHTM, UID/04413/2020), the Research Centre for Endogenous Resource Valorization (VALORIZA, UIDB/05064/2020), the Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB, UIDB/04033/2020) and the Centre for Natural Resources, Environment and Society (CERNAS, UIDB/00681/2020). This work was supported by national funds, through Fundação para a Ciência e Tecnologia (FCT, Portuguese Foundation for Science and Technology), under projects UIDB/04750/2020 and LA/P/0064/2020.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board Comissão de Ética para a Saúde CHSJ; reference number: 99/2015).