Knowledge and practices on consumption of free-range chickens in selected rural communities of KwaZulu-Natal, South Africa, with focus on zoonotic transmission of Toxoplasma gondii and Toxocara spp.

Chickens are a host to a variety of pathogens of zoonotic importance and this depends more on the husbandry system practiced. Toxoplasma gondii and Toxocara spp which are more prevalent in free-range chickens (FRC) can be acquired by humans via the ingestion of raw or undercooked meat (muscle) and/or viscera contaminated with infective stages of T. gondii and Toxocara spp. This study aimed to assess knowledge and practices on the household consumption of FRC meat and viscera by rural communities in KwaZulu-Natal (KZN) province, South Africa, as a risk factor in the transmission of zoonotic pathogens with special emphasis on T. gondii and Toxocara spp. A cross-sectional study was conducted on twenty (20) randomly selected households in four selected communities located on the northern coast (Gingindlovu and Ozwathini) and southern coast (uMzinto and Shongweni) of KZN province using a semi-structured questionnaire. To determine the presence of selected zoonotic pathogens in FRC, birds were purchased from randomly selected households in the study localities for sacrifice. Brain tissues were collected and subjected to molecular detection of T. gondii using TOX4 and TOX5 primers while other tissues and organs that were collected were subjected to molecular detection of Toxocara spp using Nem 18S primers. Questionnaire data were analyzed using the statistical package for social sciences (SPSS) version 25.0. Descriptive and chi-square statistics were used to assess knowledge and practices related to FRC consumption and zoonosis transmission. Molecular results showed four positive samples for T. canis from Gingindlovu (n = 1), uMzinto (n = 1), and Shongweni (n = 2). The role of FRC consumption in zoonosis transmission is discussed.


Introduction
Chickens are major agents in parasite transmission due to their exposure to infective stages of parasites existing in a contaminated environment (Javaregowda et al. 2016;De Vries et al. 2018). Commonly reported zoonotic parasites from chickens include but are not limited to Toxoplasma gondii and Toxocara spp (Zibaei et al. 2017;dos Santos Silva et al. 2020). Toxoplasma gondii is an apicomplexan parasite that causes toxoplasmosis while Toxocara spp are responsible for toxocariasis in animals and humans globally (Dubey 2020). The main definitive hosts of Toxoplasma gondii are felids, while birds and a wide range of animals serve as intermediate hosts (Gaulin et al. 2020). Toxoplasmosis and toxocariasis are neglected infections of zoonotic significance in animals and humans that are underreported due to lack of surveillance (Schurer et al. 2016;Etter et al. 2019). Toxoplasmosis causes production losses in animals resulting in huge economic losses in livestock industry. It can result in abortions mainly in primiparous women infected during pregnancy and hydrocephalus in infants as well as causing fatal diseases such as encephalitis in immunocompromised people (Etter et al. 2019). Approximately one-third of humanity has been exposed to Toxoplasma gondii (Dubey 2020). Although Toxoplasma infections are reportedly mild in South Africa due to the low virulence of the local strains (Jacobs, 1977), toxoplasmosis has been reported in Johannesburg, the Free State, the Western and Eastern Cape, and Kwazulu-Natal (Sonnenberg et al. 1998;Khabisi 2001).
Toxocara canis and T. cati are helminth parasites of canids and felids; they also are responsible for human toxocariasis worldwide (Yoshida et al. 2016), and a variety of paratenic hosts are involved which include chickens and especially free-range chickens (FRC) due to the husbandry practices which allow them to have contact with contaminated environments (Zibaei et al. 2017). According to the extrapolation of the world population in 2016, about 1.4 (1.2-1.5) billion individuals worldwide were estimated to be exposed to Toxocara (Rostami et al. 2019). The public health and economic importance of toxocariasis are greatest in the tropics and subtropics, where it is estimated that more than 1 billion people are infected with parasitic helminths (Ziegler and Macpherson 2019). Previous studies have reported the prevalence of Toxocara spp in animals and the seroprevalence of T. gondii in animals and humans in South Africa; however, studies on molecular identification of these parasites in food animals are limited in South Africa. Hence, this study aimed at molecular identification of T. gondii and Toxocara spp in free-range chickens in South Africa.
Although FRC contributes significantly as an affordable source of animal protein, information on their role in the transmission of zoonotic pathogens is scanty (Rodrigues et al. 2019). They are normally infected during scavenging when they ingest the infective stage of various species of parasites from a contaminated environment (Sasse et al. 2020). Humans may acquire infection indirectly via the consumption of raw or undercooked infected chicken viscera or meat containing, for example, tissue cysts of T. gondii or larvae of Toxocara spp from canids and felids (Fan et al. 2015;Gaulin et al. 2020). Once ingested by humans, the parasites depending on the species migrate through the viscera and are deposited in various organs where they cause varying degrees of symptoms ranging from fever, headache, sore throat, arthralgia, myalgia, and blindness depending on the affected organs, infection intensity and duration, host age, and immunity status of the infected host (Holland and Hamilton 2013;Fan et al. 2015;Gaulin et al. 2020). Consumption of FRC viscera or meat is a dietary habit common in different resource-poor rural communities worldwide and dependent on socio-cultural practices and culinary habits where it can either be eaten raw or undercooked (Broglia and Kapel 2011). The practice of eating raw or undercooked viscera or meat is associated with zoonosis transmission (Trevisan et al. 2019), for instance, cases of toxocariasis transmission have been reported after ingesting raw chicken liver (Nagakura et al. 1989;Morimatsu et al. 2006;Camposda-Silva et al. 2015). Similarly, human toxoplasmosis outbreaks have been reported among individuals who consumed raw or undercooked meat (Dawson 2005;Choi et al. 1997).
In South Africa, the greater population lives in rural areas and rears chickens following a free-range system (Mwale and Masika 2009;Mukaratirwa and Khumalo 2010;Malatji et al. 2016). In the KwaZulu-Natal (KZN) province of South Africa, the majority of the population are rural livestock farmers and rear FRC for consumption, marketing, and socio-cultural purposes (Naidoo 2005). The farming practice allows chickens to scavenge freely in the environment during the daytime and use trees for shelter at night or be confined to rustic chicken runs (Naidoo 2005).
Furthermore, there is an increased possibility of FRC ingesting infective oocysts and/or eggs of T. gondii and Toxocara spp respectively in the environment frequented by stray cats and dogs during scavenging in the KwaZulu-Natal (KZN) province (Tannent et al. 2010;Mukaratirwa and Singh 2010). Cats are the definitive hosts of T. gondii while cats and dogs are definitive hosts of Toxocara cati and Toxocara canis respectively. The occurrence of these stray definitive hosts in the province may lead to persistent environmental contamination with these parasites (Mukaratirwa and Singh 2010;Szwabe and Błaszkowska 2017).
Moreover, change in globalization has led to the adoption of a variety of culinary and consumption patterns regarding raw or undercooked food as delicacies (Broglia and Kapel 2011). Besides, due to the high poverty level in rural areas of KZN, there is a high level of household food insecurity thereby leading to alternative foods and various ways of food preparation (Tarwireyi and Fanadzo 2013). Understanding the consumption pattern of the much available FRC viscera or meat is imperative in these communities as a basis for guaranteeing food security as well as identifying possible transmission routes of food-borne diseases such as toxoplasmosis and toxocariasis.
Considering the poor socio-economic status and food insecurity of the rural communities in the KZN province of South Africa, this study aimed to determine the presence of selected zoonotic parasites in FRC and the factors related to the transmission of zoonotic pathogens through household consumption patterns of FRC viscera or meat and preparation practices in the study area.

Study design and sample size determination
A cross-sectional study was conducted in four rural communities in the KwaZulu-Natal province to assess knowledge and practices of consumption of FRC viscera or meat with a focus on T. gondii and Toxocara spp transmission from March to July 2019. Localities where the study was conducted and their population sizes are as follows: Gingindlovu (GI) (1109) and Ozwathini (OZ) (1979) on the northern coast and uMzinto (MZ) (16,205) and Shongweni (SH) (427,613) in the southern coast of KZN ( Fig. 1) (http:// www. durban. gov. za/). These localities have sugar cane farming as their main livelihood followed by livestock farming which includes rearing of FRC. The study population comprised 80 participants selected using simple random sampling where all households in each locality were given numbers which were subjected to a random selection (lottery method) of 20 households per locality. The sampling frame consisted of the number of households in each study locality and each participant selected represented a household for each locality. The sample size was calculated using the following equation with a 95% confidence level and 11% error margin; n = 1.96 2 pq/L 2 , where n = sample size, p = prevalence (0.5), q = 1-p, and L = limits of error on the prevalence.

Study procedure
After briefing the community leaders regarding the objectives of the study, 20 household representatives were randomly selected from each locality, and consent was obtained regarding their willingness to participate in the study. Questionnaires were translated from English to isiZulu, which is the local language in all the study localities, and were administered to the randomly selected participants following an interview-guided approach. Before administration, a pilot study was done to validate the tool. The questionnaire administration process took approximately 20 min for each participant. Before completion of the questionnaires, all participants gave their written informed consent to take part in the study. They were also reassured of the confidentiality of all disclosed information and that only anonymised findings will be disclosed during feedback and in written reports.
Data collected from the interview included socio-demographic information, knowledge, and practice of participants related to the preparation and consumption of FRC meat and viscera. Questions were asked specifically on habits related to the consumption of chicken meat and viscera, the preferred method of preparation, and the designated members of the family who eat each type of viscera. The demographic information of the participants interviewed included age, gender, household size, educational qualifications, and occupation of respondents. Information on ownership of FRC including the number of FRC owned per household was also collected.

Collection of samples from free-range chickens
Forty-two FRC were randomly purchased from households owning chickens on a willing seller basis in four selected rural communities in the Northern [Gingindlovu (GI), Ozwathini (OZ)], and Southern Coasts [uMzinto (MZ), Shongweni (SH)], of the KZN province. Chickens selected for the study were euthanized by decapitation according to guidelines approved by Animal Ethics of the University of KwaZulu-Natal South Africa. Tissue from various parts of each chicken such as the brain, heart, spleen, lungs, liver, kidney, crop, duodenum, intestines, thigh, breast, and pectoral were collected, digested, and examined for Toxocara larvae using the modified acid/pepsin digestion (Zibaei et al. 2017). The digests were washed and filtered through a sieve with 200/125/20-µm apertures. Collected Toxocara larvae were kept in 70% ethanol until DNA extraction. Brain samples for molecular detection of T. gondii were collected as previously described by Carlos and Jack 1998. Briefly, after euthanization, the skull was opened with a pair of small, curved scissors to expose the brain. The brain was then removed with forceps and carefully preserved in 70% ethanol.

Molecular identification
The retrieved nematode larvae from each of the chicken samples were subjected to molecular analysis using a QIAamp DNA Mini Kit (Qiagen Inc.) and used in subsequent PCR reactions. PCR reactions for the amplification of 18S rRNA were performed with nematode-specific primers Nem_18S_F (CGC GAA TRG CTC ATT ACA ACAGC (23 bases) and Nem_18S_R (GGG CGG TAT CTG ATC GCC (18 bases). A standard reaction volume was 20 µL, comprising the following: NEB OneTaq 2X MasterMix with Standard Buffer at 10 µl, primers (10 µM) at 1 µl each, and nuclease-free water at 7 µl were added. To each reaction, 1 µl of extracted nematode DNA template was added, typically containing around 10-30 ng/µl of genomic DNA. The PCR conditions for the amplification of 18S rRNA were as follows: initial denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 30 s, primer annealing at 50 °C for 30 s, and extension at 68 °C for 1 min. The final extension was at 68 °C for 10 min. The integrity of the PCR amplicons was visualized on a 1% agarose gel. Thirty brain samples (halves) were tested for Toxoplasma gondii. DNA was extracted from half of the brain tissue of chickens using a commercial kit. PCR on DNA extracts was done using the primer, TOX4 (5' -CGC TGC AGG-GAG GAA GAC GAA AGTTG-3') and TOX5 (5'-CGC TGC AGA CAC AGT GCA TCT GGA T-T-3') targeting the 529 bp fragment. PCR was conducted according to the described protocol (Homan et al. 2000). The cycling protocol for the T. gondii DNA amplification included an initial cycle of 94 °C for 7 min (initial denaturation), followed by 35 cycles of 94 °C for 1 min (denaturation), 60 °C for 1 min (annealing), and 72 °C for 1 min (extension), followed by a final extension 72 °C for 10 min. PCR products were analyzed on 2% agarose gel by electrophoresis for 25 min at 120 V.

Statistical analysis
Data were processed and analyzed using the statistical package for social sciences (SPSS) version 25.0. Descriptive and chi-square statistics were used to assess knowledge and practices related to the consumption of FRC meat and viscera and awareness of the zoonotic transmission of T. gondii and Toxocara spp. A p-value < 0.05 was considered statistically significant. Table 1 shows the demographic characteristics of participants in all four study localities. Participants interviewed in the four localities ranged in the category of father, mother, and household member greater than 18 years. Overall, the mean age of the respondents was (47.11 ± 18.02) ( Table 1). Ozwathini (OZ) had the highest mean age (53.3 ± 16.82), followed by Shongweni (SH) (50.25 ± 17.12, GI (43 ± 18.98) and uMzinto (MZ) (41.9 ± 17.80). A significant difference was observed between the educational level of study respondents among study locations (p < 0.05). Most respondents (47.5%, 38/80) had a high school education while only (20%, 16/80) had completed tertiary education. The percentage of respondents who had tertiary education was highest in OZ (40%, 8/20), followed by GI (30%, 6/20), and SH (10%, 2/20), while none of the respondents in MZ had tertiary education.

Knowledge of zoonosis transmission associated with the consumption of FRC viscera
Overall, knowledge of zoonotic disease (mainly related to toxoplasmosis and toxocariasis) transmission associated with the consumption of raw or undercooked FRC meat and viscera in the study localities was estimated at 31.3%. There were no significant associations found between knowledge and considered variables. Knowledge did not vary among the localities (35%, 7/20) in MZ and SH followed by GI (30%, 6/20) and OZ (25%, 5/20) ( Table 2). The proportion of respondents knowing zoonosis transmission through consumption of raw/undercooked chicken viscera was high in the age group 41-50 years and highest in GI (20%, 4/20) followed by SH (15%, 3/20) and OZ (10%, 2/20), while in MZ, it was highest in the age group ≥ 61 (20%, 4/20). This difference was, however, not statistically significant (p > 0.05).

Molecular results
All the 30 brain samples were negative for T. gondii. Toxocara canis larvae were detected and confirmed through sequencing in the right pectoral muscle, liver, left thigh, and lungs of FRC from Gingindlovu (GI) (n = 1), uMzinto (MZ) (n = 1), and Shongweni (SH) (n = 2). There were no Toxocara larvae found in FRC from Ozwathini (OZ). The summary of molecular results is shown in  (Table 5).

Discussion
Several studies have reported the vulnerability of resourcepoor communities who keep FRC to zoonotic diseases such as toxoplasmosis and toxocariasis (Neghina 2010;Santarém et al. 2011;Mirza and Rathore 2019). Socio-demographic factors have been reported to influence the food and meatgathering practices of people in a way that predisposes them to parasitic infections (Simeone 2008;Drescher et al. 2012;Goyette et al. 2014). Our study showed that a quarter of the respondents in the study areas had only completed primary education. This is consistent with reports from other rural   (Mwale and Masika 2009;Spaull 2015). Also, this study showed that the overall percentage of zoonosis transmission awareness was comparable to 30.1% recorded from cattle farmers in Senegal (Tebug et al. 2015) but higher than the awareness level of 19.1% of the zoonotic risk associated with livestock in Ibadan Nigeria (Awosanya and Akande 2015). However, it was lower than the 69% and 79.74% that were reported in Cambodia and Punjab respectively (Osbjer et al. 2015;Singh et al. 2019). Although no significant associations were found between knowledge of zoonosis transmission and considered variables in all the localities, the high level of knowledge obtained among respondents with high school education disagrees with the report from Western Ethiopia where KAP scores were higher among people with tertiary education (Tamiru et al. 2022). This can be attributed to the fact that the majority (45%) of the respondents in this study have a high school education. Similarly, regarding occupation, the higher level of knowledge observed among unemployed respondents disagrees with the report from Western Ethiopia where good KAP scores were recorded among people with good job types (Tamiru et al. 2022). This can be attributed to the large percentage of unemployed (82.5%) constituting the respondents. A similar explanation is responsible for the high knowledge of zoonosis transmission observed among women in the study locations where most of the respondents (61.3%) were women.
Furthermore, this study revealed that ownership of FRC in the study locations was 65% (52/80), which is higher than the 57.7% (41/71) reported in Ethiopia (Sambo et al. 2015) but lower than the 93.5% and 84% poultry (duck and chicken) ownership observed in the Eastern Cape province of South Africa and Cambodia respectively (Mwale and Masika 2009;Osbjer et al. 2015). Also, the average flock size (17.2 ± 1.4) observed in this study is higher than (16 ± 2.1), reported in the Eastern Cape province of South Africa (Mwale and Masika 2009), but lower than (22.03 ± 2.85) in Limpopo province and (28.40 ± 2.57) earlier reported in the KwaZulu-Natal province respectively (Malatji et al. 2016). Regarding consumption patterns, the majority (76.3%, 61/80) of respondents reported the practice of consumption of FRC viscera in their households. The reason for the high demand for chicken viscera in the study area is however unknown. Studies have identified the role of poor socio-economic factors as well as globalization as important factors in meat consumption patterns (Tambi 2001;Simeone 2008;Goyette et al. 2014;Robertson et al. 2014). Additionally, the viscera of chicken and other avian animals have been reported to be rich in essential nutrients for humans (Schönfeldt and Gibson 2008).
Chickens are used as sentinel agents in monitoring the prevalence of infections in the environment due to their ground-feeding habits (Dubey et al. 2005). Molecular techniques employing non-coding 529 base pairs DNA fragment and the internal transcribed spacer 1 (ITS-1) of the rRNA gene have proved effective in the identification of T. gondii and several organisms to species level due to the variations of the (ITS-1) of the rDNA (Santos et al. 2010), resulting in a higher detection rate (Chemoh et al. 2016). Also, PCR has been used unequivocally for the detection of animal and human toxocariasis (Dewair and Bessat 2020).
This study is the first to consider the prevalence of T. gondii in free-range chickens from the KZN province, South Africa, using a molecular approach. The absence of T. gondii observed in the brain tissues of chicken in this study might be an indication that the FRC sampled in our study may have not been exposed to T. gondii oocysts. This is consistent with a report from a study conducted on retail turkey meat products where T. gondii DNA was not detectable using magneticcapture PCR (Koethe et al. 2015). Another study reported a low prevalence of T. gondii infection in feral rodents and insectivores (Meerburg et al. 2012). The absence of T. gondii in this study may be attributed to the non-survival of T. gondii oocysts in the environment due to hot and dry temperatures (Lukášová et al. 2017). Toxoplasma gondii infections thrive in mild temperature climates than in a hot and dry environment (Dubey 1998;Gilot-Fromont et al. 2012) or dry and very cold winter (Smallbone et al. 2017). For instance, T. gondii oocysts survived for 32 days at 35 °C, 9 days at 40 °C, and only 1 day at 45 °C (Dubey 1998). The chickens used in this study were obtained between March and July which is usually characterized by the highest temperature and lowest temperature respectively (Masemola et al. 2020). The mean daily minimum temperature in KZN is around 35 °C in March and 16.76 °C in July (Dzikiti et al. 2022).
On the other hand, the presence of T. canis observed in this study agrees with Zibaei et al. (2017) who isolated Toxocara canis larvae from the liver, skeletal muscles, duodenum, and brain of broiler chickens and Okada et al. (2021) who reported the occurrence of T. cati and T. tanuki from the thigh and breast meat from chicken, respectively. Similarly, Davidson et al. (2012) reported T. cati from pigs at a slaughterhouse in Norway. Also, T. vitulorum was detected in bovine milk samples using a molecular approach (Dewair and Bessat 2020). The prevalence of Toxocara infection observed in this study (9.5%: 4/42) is consistent with reports from other studies where low prevalence has been reported. Zibaei et al. (2017) reported 15.2% (5/33) from broiler chickens while Okada et al. (2021) reported 4% (2/50) from culled chickens from a commercial farm and Davidson et al. (2012) reported a prevalence of 1% (1/100) from a pig in a slaughterhouse from Norway. The occurrence of T. canis in FRC observed in this study indicates that the chickens are being exposed to environments contaminated with T. canis eggs from dogs in the localities studied. Considering the high rate of consumption of chicken viscera practiced by communities in this study, although most preferred "well-cooked," it is important to create awareness of the role of FRC as paratenic hosts of important zoonotic parasites of dogs and cats such as T. gondii and Toxocara spp. Furthermore, the application of the PCR technique used in this study could be employed in routine detection methods for Toxocara larvae in organs of suspected infected animals, especially in toxocariasis endemic areas.
We also recommend the participation of all stakeholders through a One Health approach in designing control and prevention strategies for zoonotic pathogens affecting these communities using the findings from this study as a basis.