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. 2024 Aug 14;6:100207. doi: 10.1016/j.crpvbd.2024.100207

Isolation and genetic characterization of Toxoplasma gondii from chickens from public markets in Pernambuco State, Brazil

Renato Amorim da Silva a, Raissa Santana Renovato a, Hannah Tsuruzaki Kirzner de Barros e Silva a, Maria Luiza Didier Marques a, Pollyanne Raysa Fernandes Oliveira a, Jéssica de Crasto Souza Carvalho-Reis a, Paul M Bartley b, Frank Katzer b, Érika Fernanda Torres Samico-Fernandes a, Renata Pimentel Bandeira de Melo a,⁎, Rinaldo Aparecido Mota a
PMCID: PMC11381444  PMID: 39253292

Abstract

This study aimed to evaluate the presence and viability of Toxoplasma gondii in chickens intended for human consumption in the Pernambuco State, Brazil. Blood and tissue samples were collected from 25 chickens sold in markets in Recife, Pernambuco. Samples were evaluated by indirect immunofluorescence assay (IFA) to detect antibodies to T. gondii. Pools of brain and heart of seropositive chickens were subjected to bioassay in two Swiss Webster mice, which were evaluated for 45 days then tested by IFA to detect seroconversion. The mice were euthanized, and their brains were evaluated for cysts. Peritoneal lavage was also conducted in mice that exhibited clinical signs. Brains containing cysts or peritoneal lavage with tachyzoites were inoculated into MA-104 cells. Brains of mice inoculated with the same tissue were pooled and analysed by ITS1-PCR. We obtained a frequency of antibodies to T. gondii of 68.00% (17/25) in chickens, and a seroconversion rate of 70.58% (24/34) in mice. Detection of Toxoplasma ITS1 DNA confirmed an isolation rate of 41.1% (7/17). Three isolates were characterized by mnPCR-RFLP as genotypes ToxoDB#36 and ToxoDB#114. We highlight the occurrence of ToxoDB#36 in chickens in Pernambuco State and the parasitesʼ viability in chickens intended for human consumption.

Keywords: Toxoplasma gondii, Chicken, Mouse bioassay, PCR-RFLP, Public health, Zoonosis

Graphical abstract

Image 1

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Highlights

  • •

    Toxoplasma gondii was detected in chickens intended for consumption with a frequency of 68.0%.

  • •

    Seven T. gondii isolates were obtained by mouse bioassay using chicken tissues.

  • •

    Genotype ToxoDB #36, previously associated with human congenital toxoplasmosis, and Genotype #114 were identified.

  • •

    Finding viable T. gondii in chicken intended for human consumption poses a risk.

1. Introduction

Industrial poultry farming is responsible for 98% of the chicken supplied in Brazil. However, the consumer market is increasingly demanding organic/free-range products for their health benefits. Free-range chicken farming stands out for producing meat with a texture, appearance and flavour that is more pleasing to the consumer, free of antibiotics or growth promoters and prioritizing animal well-being (Cavalcanti, 2019). There are however many issues that arise during food production limiting the expected quality of products by consumers, in which poor sanitation is one of the main causes of low production and poor economic performance in broilers (Aquino et al., 2020).

Toxoplasma gondii is a protozoan with worldwide distribution and it is speculated that at least one-third of the human population is infected, with varying prevalence depending on the geographical region (Molan et al., 2019). Chickens (Gallus gallus domesticus) usually do not present clinical signs of toxoplasmosis; however, they pose a transmission risk to humans when their meat or organs are consumed when not properly cooked (Dubey, 2010). Also, chickens are used as an indirect indicator of environmental contamination with T. gondii oocysts, as they are frequently exposed to this parasite through their soil-scratching/pecking habits (Dubey, 2010). As a consequence of the low level of technification in extensive (backyard) farming, chickens are routinely exposed to a contaminated environment (Schares et al., 2017).

Population structure studies of T. gondii try to elucidate its genetic variability, providing epidemiological insights into transmission and clinical manifestations of the disease (Shwab et al., 2014). Data obtained from South American isolates, mainly from Brazil, showcase a population of T. gondii with high genetic diversity and a predominance of non-clonal genotypes (recombinants or atypical). This diversity originates from the parasite's sexual recombination, favoured by the wide biodiversity present in Brazil (Costa et al., 2021). Studies conducted in Brazil highlight the genetic diversity of T. gondii found in chickens of the Northeast Region, with greater occurrence of the genotypes #13, #59, #146 and #163 (Feitosa et al., 2016; Costa et al., 2021).

This study aimed to isolate and identify T. gondii genotypes from chickens destined for human consumption sold in public markets in Pernambuco State, Brazil, to provide data on the risk this meat poses for public health.

2. Materials and methods

2.1. Sample collection

Blood and tissue samples were collected from 25 chickens reared extensively, on small farms (considered as backyard production) located in the regions Zona da Mata and Agreste, and sold in public markets in Recife, capital of Pernambuco State, between December 2022 and May 2023. The chickens were slaughtered at the markets’ slaughterhouse after they were sold. Blood and tissue samples were collected and processed at the slaughterhouse.

Blood samples were centrifuged at 1500× g for 10 min to obtain the serum, which was stored at −20 °C until serological diagnosis. The brain and heart samples were stored at 4 °C until processing, which was carried out within 24 h after collection.

2.2. Serological diagnosis and bioassay in mice

Serological detection of antibodies to T. gondii in the chickens was performed by indirect immunofluorescence assay (IFA), as established by Camargo (1964). Tachyzoites of the T. gondii ME49 strain were used as antigen (concentration of 1200–1500 tachyzoites/μl). Serum samples, as well as known positive and negative controls were diluted 1:16 in PBS and slides were incubated at 37 °C for 30 min. The slides were then incubated with a rabbit anti-chicken Ig antibody – FITC (Sigma-Aldrich, St. Louis, MO, USA) diluted in PBS with 0.02% Evans blue at 37 °C for 30 min, then washed with PBS. Samples were evaluated using an epifluorescence microscope (Olympus BX60-FLA, USA) and considered positive when whole peripheral surface fluorescence showed in at least 50% of the tachyzoites in the well.

The tissue samples of seropositive animals were submitted for bioassay in mice utilizing the pepsin digestion protocol (Dubey, 1998). Pooled samples of brain and heart (10 g) of each chicken were macerated, suspended with 50 ml of 0.9% NaCl solution, and then digested by addition of 50 ml of an acid-pepsin solution (2.6 g of pepsin 1:10,000; Dinâmica Química Comtemporânea LTDA), 7 ml of hydrochloric acid (12N), and 5.0 g of NaCl added in 500 ml of sterile distilled water (pH 1.1–1.3) in an incubating shaker at 37 °C for 60 min. Samples were then filtered through gauze, centrifuged at 1500× g for 10 min, and had the pH neutralized with 7 ml of a 1.2% sodium bicarbonate (pH 8.3) solution before being resuspended with 43 ml of PBS and centrifuged for 1500× g for 10 min. The pellet was then resuspended with 5 ml of PBS solution with the addition of 100 IU/ml penicillin and 100 μg/ml streptomycin. After digestion, 0.5 ml of each sample was inoculated into two Swiss Webster mice (Immunopathology Bioterium Keizo Asami of the Federal University of Pernambuco - LIKA/UFPE) by intraperitoneal administration.

Animals were monitored at least once daily for a period of 45 days, during which signs of illness such as raised hair, abdominal pain, ocular photosensitivity, cyanosis, and signs of neuropathology (head turning, opisthotonos or lateralization) were recorded. Clinical signs were classified as minimal (raised hair), mild (mild abdominal pain, characterized by slight flank retraction), and moderate (noticeable flank retraction, cyanosis, ophthalmic signs or neurological signs). To limit animal suffering, a humane endpoint was established using clinical signs previously described in the mice. Animals with moderate clinical signs were immediately euthanized. At 30 days post inoculation (dpi) blood samples were collected from the mice by puncture of the submandibular vein (Golde et al., 2005) and serum samples were obtained to evaluate seroconversion (IgG antibodies to T. gondii) by IFA at a dilution of 1:16. On 45 dpi mice were euthanized, and the brains were collected and evaluated for the presence of tissue cysts by brain fragment imprinting on microscope slides and direct visualization by optical microscopy. Peritoneal lavage was collected from mice with clinical signs of infection. The same procedures were carried out in mice that had to be euthanized prior to the end of the experiment. We considered successful isolation in cases when the presence of T. gondii genetic material was observed in mice brain or peritoneal lavage (Dubey, 2010).

2.3. Cell culture

The brains of mice which seroconverted (positive IFA), as well as produced peritoneal lavage samples with structures suggestive of tachyzoites were inoculated onto layers of MA-104 cells (ATCC® CRL-2378.1) for in vitro maintenance. Cultures were incubated at 37 °C in a humidified 5% CO2 atmosphere in Dulbeccoʼs Modified Eagle Medium (DMEM) supplemented with 10% horse serum and the flasks were evaluated daily for a 21-day period. The flasks containing structures suggestive of tachyzoites, cellular lysis or parasitophorus vacuoles underwent T. gondii-specific ITS1 PCR. Confirmed isolates were then cryopreserved using 20% dimethyl sulfoxide (DMSO) diluted in horse serum, aliquoted into 1.5 ml cryogenic vials, and stored in a liquid nitrogen container (−196 °C).

2.4. DNA extraction and PCR

DNA was extracted from 25 mg of mice brain samples, 100 μl of resuspended mice peritoneal lavage pellets, and 100 μl of resuspended cell culture pellet using the Wizard DNA Genomic Purification Kit (Promega, Madison, WI, USA), according to the manufacturerʼs instructions. DNA extraction of brain tissue and peritoneal lavage was carried out on pooled samples of the mice inoculated in duplicate.

Molecular identification of T. gondii DNA was carried out by ITS1 Nested-PCR (Hurtado et al., 2001). Amplified products were analysed by agarose gel (2%) electrophoresis stained with BlueGreen (LGC®) and visualized under UV light. DNA extracted from ME49 tachyzoites was used as a positive control and DNA-free water was used as a negative control. Samples, which tested positive for T. gondii using the ITS1 PCR, were genotyped.

2.5. Genotyping and phylogeny

Determination of the T. gondii genotype was conducted using a multiplex nested PCR-restriction fragment length polymorphism (mnPCR-RFLP) method using 10 genetic loci (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico), according to Su and Dubey (2020) with modifications described by Feitosa et al. (2024). Digested products were then analyzed by agarose electrophoresis, containing Gel Red (1:10000 dilution) (Biotium, Fremont, CA, USA) and visualized under UV light. Genotypes were determined according to the ToxoDB DataBase (http://toxodb.org/toxo/). Control strains were S48 (Type I), M4 (Type II variant) and NED (Type III).

For phylogenetic analysis, the PCR-RFLP patterns were converted into binary data (“0” absence of band; “1” presence of band) and tabulated, according to markers, in the phylogeny reconstruction program SplitsTree4 (Huson and Bryant, 2006).

3. Results and discussion

Frequence of IgG antibodies to T. gondii was 68.0% (17/25) in chickens. On 30 dpi, 70.5% (24/34) of the mice had seroconverted, as shown in Table 1. The frequency of antibodies found in this study is high when compared to other studies carried out on free-range (extensively reared) chickens in the State of Pernambuco, such as Sá et al. (2017) who obtained a prevalence of 27.9% (176/629) and Fernandes et al. (2016) who detected a prevalence of 40.5% (86/212).

Table 1.

Toxoplasma gondii isolation from chickens intended for human consumption: seroconversion of mice, and molecular detection in mice samples (brain and peritoneal lavage) and cell culture.

Sample No. of seroconverted mice ITS1 PCR result
Peritoneal lavage Brain Cell culture
G01 2 – + –
G02 0 – NP NP
G04 2 – – –
G05 0 – NP NP
G06 0a + NP +
G08 0 – NP NP
G09 2 – – –
G10 2 – + –
G13 2 – + –
G14 2 NP + +
G15 2 – + –
G16 2 – – –
G17 2 – – –
G19 2 – – –
G21 0 – NP NP
G22 2 – + –
G25 2 – – –
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Key: , negative; +, positive; NP, not performed.

a

Mice inoculated with sample G06 presented hyperacute symptoms and were euthanized before IgG seroconversion; tachyzoites were observed in peritoneal lavage of both mice.

It is known that extensive farming practices lead to greater exposure of chickens to infectious agents, including T. gondii (Abbaszadeh et al., 2022), compared to chickens raised in intensive systems, which usually report a lower prevalence of disease due to greater health control and lack of contact between birds and the environment (Dubey et al., 2020). The detection of antibodies to T. gondii in chickens confirms exposure of the host to the parasite; however, it does not confirm the existence of viable tissue cysts able to cause infection and seroconversion in susceptible hosts (Dubey et al., 2020). This may explain why not all of the mice seroconverted following inoculation with tissues from seropositive birds (Table 1). In this study, all samples were processed within 24 h after slaughtering, therefore, the post-mortem time probably had no impact on the effectiveness of isolation (Dubey et al., 2020).

Clinical signs such as raised hairs, abdominal pain and/or ophthalmic signs, such as photosensitivity, were observed in most mice (32/34), except for mice inoculated with sample G14 (TgCkBrPe3), which did not present any clinical sign of infection. At 5 dpi, mice inoculated with sample G06 (TgCkBrPe1) presented hyperacute clinical signs and were euthanized to limit suffering. The mice inoculated with sample G06 did not seroconvert likely due to insufficient time, as these mice were culled on 5 dpi and previous studies verified the detection of IgG antibodies in experimentally infected mice from 10 dpi onwards (Quan et al., 2009). However, structures suggestive of tachyzoites were observed in peritoneal lavages of the two mice, and a pool of the lavages was directly inoculated in a culture of MA-104 cells.

On 45 dpi, tissue cysts were observed in one (1/34) of the inoculated mice. The ITS1 PCR was positive in 50.0% (6/12) of mouse brain pools and 6.2% (1/16) of peritoneal lavage samples, confirming T. gondii infection, which represents an isolation rate of 41.1% (7/17). Two of the seven confirmed T. gondii isolates were successfully maintained in MA-104 cells and cryopreserved in liquid nitrogen. The presence of viable T. gondii in chicken tissues demonstrates a potential health risk to the population that consumes this meat.

The habit of scratching and pecking makes chickens an indicator of environmental contamination with T. gondii oocysts (Dubey, 2010). Furthermore, the quality of water and food offered also directly affects the occurrence of T. gondii infection (Shokri et al., 2017). Therefore, the presence of the viable parasite in the farming environment of these birds can be inferred. Previous studies have also stated that there is a direct relationship between the prevalence of antibodies to T. gondii in chickens and the occurrence of toxoplasmosis in the human population (Moré et al., 2012).

DNA samples, which were PCR-positive, were subjected to mnPCR-RFLP for genotypic characterization. Two T. gondii isolates (TgCkBrPE1 (G06) and TgCkBrPE2 (G14)) were fully genotyped, identifying as genotypes ToxoDB #36 and ToxoDB #114 respectively (Table 2). A third isolate (TgCkBrPE3) (G13) was partially characterized, as one genetic marker (PK1) was not amplified, though, the genetic profile of the other markers suggests that this isolate may be genotype #114, like the TgCkBrPE2 isolate obtained in this study. Noteworthy is the fact that if isolate TgCkBrPE3 is not allele I at the PK1 locus, it may be a new genotype that has not been previously described.

Table 2.

Genetic characterization by mnPCR-RFLP of Toxoplasma gondii isolates obtained from chickens intended for human consumption in Pernambuco State, Brazil.

Sample Isolate Genetic markers
 ToxoDB - RFLP genotype
SAG1 5′-3′ SAG2 Alt.SAG2 SAG3 BTUB GRA6 c22-8 c29-2 L358 PK1 Apico
G06 TgCkBrPE1 I I I III I III II I III I III #36
G14 TgCkBrPE2 I III III I III III III I III I I #114
G13 TgCkBrPE3 I III III I III III III I III NA I Atypical
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Abbreviation: NA, not amplified.

The genotype #36 has previously been reported in chickens in other Brazilian states including Espírito Santo (Ferreira et al., 2018), Bahia (Rocha et al., 2018) and Rio de Janeiro (Dubey et al., 2020). It was also reported in the state of Minas Gerais in a case of human congenital toxoplasmosis (TgCTBr18) and classified as having an intermediate virulence (Carneiro et al., 2013). Until the present study, there are no reports of the occurrence of genotype #36 in the State of Pernambuco. The genotype #114 was reported in free-ranging chickens in the State of Pernambuco (Shwab et al., 2014).

From the obtained genotypes it was possible to construct a phylogenetic tree, in which reference strains were compared (clonal lineages I, II and III; BrI, BrII, BrIII, BrIV, and genotypes found in chickens and other species in Brazil). Fig. 1 shows the tree drawn based on the isolates obtained in the present study compared to previously identified genotypes, where TgCkBrPE1 (ToxoDB#36) was closely related to genotypes #13 and #143. Furthermore, there was a proximity to the type I clonal lineage and TgCkBrPE2. The isolate TgCkBrPE2 (ToxoDB #114) was phylogenetically closer to genotype #279 and was closely related to the Type clonal lineage III. Both isolates #36 and #114 were also close to other genotypes recorded in neighbouring states such as #13, #143 and #279. It is important to highlight that genotype #13 has already been described with great frequency in different regions of Brazil, in production animals (sheep, goat, pig, and chickens) and wild species (Almeida et al., 2017; Feitosa et al., 2017, 2024).

Fig. 1.

Fig. 1

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Phylogenetic analysis of Toxoplasma gondii isolates obtained in the present study (red) and characterized by RFLP-PCR utilizing the clonal lineages I, II, III, II-variant, BrI, BrII, BrIII, BrIV, and isolates obtained from chickens in Brazil as reference strains.

4. Conclusions

The present results demonstrate a high prevalence of viable Toxoplasma gondii in chickens sold in popular markets and intended for human consumption in the State of Pernambuco, Brazil. These results play an important role in the epidemiology of toxoplasmosis in human health and other animal species that live in the same environment, or that may be fed with the carcasses of these birds. The presence of atypical genotypes can be attributed to the genetic diversity reported in the State of Pernambuco. Finding viable ToxoDB#36 in a chicken, which was previously detected in a human case of congenital toxoplasmosis, highlights the risk these atypical genotypes pose to human health.

Funding

This study was supported by the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) which provided a scholarship to Renato Amorim da Silva (grant number: 0180.5.05/22). Paul Bartley and Frank Katzer were supported by funding from the Scottish Government through the Rural and Environment Science and Analytical Services (RESAS) Strategic Research Programme 2022–2027, project number MRI-B6-1: “Addressing knowledge gaps in the sources, epidemiology and genetic diversity of important foodborne pathogens”.

Ethical approval

The experiment conducted in this study was approved and carried out following the recommendations of the Ethics Committee on Animal Use (CEUA) of Federal Rural University of Pernambuco - UFRPE (protocol number 1584150622) and was carried in accordance with the ethical principles of animal experimentation adopted by the Brazilian College of Animal Experimentation (COBEA).

CRediT authorship contribution statement

Renato Amorim da Silva: Formal analysis, Investigation, Data curation, Writing – original draft, Visualization. Raissa Santana Renovato: Investigation. Hannah Tsuruzaki Kirzner de Barros e Silva: Investigation. Maria Luiza Didier Marques: Investigation. Pollyanne Raysa Fernandes Oliveira: Investigation, Data curation, Writing – review & editing. Jéssica de Crasto Souza Carvalho-Reis: Formal analysis, Investigation. Paul M. Bartley: Formal analysis, Investigation, Writing – review & editing. Frank Katzer: Conceptualization, Writing – review & editing. Érika Fernanda Torres Samico-Fernandes: Conceptualization, Writing – review & editing. Renata Pimentel Bandeira de Melo: Supervision, Formal analysis, Data curation, Writing – original draft, Writing – review & editing. Rinaldo Aparecido Mota: Conceptualization, Supervision, Writing – review & editing, Project administration.

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Given their role as Co-Editor, Frank Katzer had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Professor Aneta Kostadinova (Editor-in-Chief).

Data availability

All data generated or analyzed during this study are included in this article.

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Associated Data

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Data Availability Statement

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