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Article

PCR Detection, Genotyping, and Differentiation of Toxoplasma gondii from Hammondia hammondi Excreted in the Feces of Cats in Poland Between 2020 and 2024

by
Dawid Jańczak
1,*,
Rafał Stryjek
2,
Aleksandra Kornelia Maj
3,
Jakub Olszewski
3 and
Olga Szaluś-Jordanow
4,*
1
Department of Infectious and Invasive Diseases and Veterinary Administration, Institute of Veterinary Medicine, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University, 87-100 Toruń, Poland
2
Institute of Psychology, Polish Academy of Sciences, 00-378 Warsaw, Poland
3
Animallab Veterinary Laboratory, 03-430 Warsaw, Poland
4
Department of Small Animal Diseases with Clinic, Institute of Veterinary Medicine, Warsaw University of Life Sciences-SGGW, 02-776 Warsaw, Poland
*
Authors to whom correspondence should be addressed.
Pathogens 2025, 14(5), 444; https://doi.org/10.3390/pathogens14050444
Submission received: 31 March 2025 / Revised: 28 April 2025 / Accepted: 30 April 2025 / Published: 30 April 2025
(This article belongs to the Section Parasitic Pathogens)
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Abstract

:
Toxoplasmosis, caused by Toxoplasma gondii, is a widespread parasitic infection affecting humans and animals. The genetic diversity of T. gondii varies across regions, with type I, II, and III strains predominantly circulating in Europe and North America. This study genotyped 67 (78.8%) T. gondii DNA isolates from cats using nested and multilocus PCR-RFLP, identifying type I, genotype #10 (ToxoDB#10), for the first time in Poland. The other 18 (21.2%) stool samples containing T. gondii-like oocysts were confirmed as Hammondia hammondi. Comparative analysis with data from other countries highlights notable regional differences in genotype prevalence. The high occurrence of genotype 3 (ToxoDB#3) in central Europe may be linked to its presence in wild rodents and insectivores, key reservoirs in the parasite’s life cycle. Additionally, genetic analysis of meat products and livestock indicates a potential transmission pathway to felines through raw or undercooked meat consumption. These findings contribute to a better understanding of T. gondii epidemiology and its implications for public health and veterinary medicine.

1. Introduction

Toxoplasma gondii is an intracellular protozoan parasite capable of infecting all warm-blooded animals, in contrast to Hammondia hammondi, which can naturally infect only a limited range of animals like mice, rats, and felids [1,2,3]. House cats and other felids are the only animals that can excrete T. gondii and H. hammondi oocysts into the environment through their feces. T. gondii oocysts become infective after sporulating for 3 to 5 days and can remain viable in water and soil for many months [4,5,6]. However, the sporulation period can be as short as one day under optimal environmental conditions, such as those found in cat litter [7]. Approximately one-third of the global human population is infected with T. gondii; however, clinical symptoms, such as fever, ocular inflammation, and lymphadenopathy, occur in fewer than 20% of infected individuals [8,9]. Toxoplasmosis poses a significant challenge in immunocompromised patients, where generalized infection may develop, leading to neurological, lymphatic, or ocular complications [10]. The primary sources of T. gondii infection in humans include raw meat, raw milk, or mature cheese containing viable tachyzoites or bradyzoites of the parasite [11,12]. Cats and other felids become infected after consuming raw meat or tissues from any warm-blooded animal that can act as an intermediate host [13]. In young cats, during their first infection, clinical signs such as diarrhea, lethargy, polydipsia, jaundice, dyspnea, and fever may be observed [14]. Infected cats can shed between 3 and 8 million oocysts during the shedding period, although in some cases, no oocysts are excreted at all [15]. Zulpo et al. confirmed that the shedding of T. gondii oocysts may remain elevated in experimentally re-infected cats even years after the primary infection. Approximately 10% and 71% of cats re-infected with different strains were found to excrete oocysts following secondary and tertiary infections, respectively [16].
Dubey demonstrated a relationship between T. gondii infection and Cystoisospora felis. In cats initially infected with T. gondii and subsequently with C. felis, reactivation of toxoplasmosis and oocyst shedding could occur. However, when the order of infection was reversed, the local intestinal immunity induced by C. felis was sufficiently strong to prevent the shedding of T. gondii oocysts. Moreover, in adult cats, a reshedding may be induced by co-infection with other infectious diseases or long-term treatment with immunosuppressive drugs [17].
The oocysts of T. gondii and Hammondia hammondi are very similar in morphology, and the parasites themselves are closely related as tissue cyst-forming coccidians [18]. However, the bioassay procedure can differentiate them. T. gondii tissue cysts are more commonly found in neural tissue, whereas H. hammondi cysts predominantly localize in skeletal muscle. Additionally, tachyzoites and bradyzoites of H. hammondii are not infective after oral administration. Moreover, H. hammondi appears to be non-pathogenic and does not cause clinical disease in cats or any naturally infected hosts, including humans [6]. Molecular techniques based on the polymerase chain reaction (PCR) are the methods for differentiating Hammondia-like organisms from T. gondii oocysts [19,20]. Due to variations in virulence among T. gondii strains, multilocus PCR-RFLP (restriction fragment length polymorphism) genotyping plays a crucial role in determining the geographic distribution and genetic diversity of clonal lineages [21,22,23,24,25]. In this study, we present the results of T. gondii and H. hammondi oocyst differentiation using PCR methods and a molecular analysis of T. gondii strains isolated from feline feces collected in Poland between 2020 and 2024.

2. Materials and Methods

2.1. Sample Collection

Between 2020 and 2024, a total of 61,648 feline fecal samples were examined using the zinc flotation method (ZnSO4, Specific Gravity = 1.31) and direct smear microscopy in a 0.9% saline solution. All stool samples were examined in a commercial veterinary laboratory as part of the diagnosis and prevention of gastrointestinal parasitic infestation. Stool consistency was evaluated in the laboratory prior to microscopic examination. Samples containing oocysts smaller than 15 μm in diameter were frozen for further molecular analysis (Figure 1).

2.2. DNA Isolation and Molecular Analysis

DNA extraction was performed using the commercial Genomic Mini AX Stool kit (A&A Biotechnology, Gdańsk, Poland). The extracted DNA was suspended in 200 μL of elution buffer and stored at −20 °C for further analysis.
To differentiate T. gondii and H. hammondii, two different PCR tests were used [19,26]. Samples positive for T. gondii were subsequently analyzed by the nested and multiplex multilocus PCR-RFLP (Mn-PCR-RFLP) using various genetic markers: the B1 gene [21,27] and SAG1, 5′SAG2, 3′SAG2, alt.SAG2, SAG3, BTUB, GRA6, Apico, C22-8, C29-2, L358, and PK1 [8,24,28,29,30,31]. The primers and restriction enzymes applied in this study are presented in Table 1 and Table 2. Both PCR and nested PCR were carried out via a MultiGene optiMAX thermal cycler (Labnet International, Inc., Taoyuan, Taiwan) using a commercial StartWarm HS-PCR Mix kit (A&A Biotechnology, Gdańsk, Poland). The 530 bp products of B1 gene amplification were sequenced and analyzed using Chromas 2.6.6 (Technelysium Pty Ltd., South Brisbane, Australia) and MEGA version 7 software. To compare the obtained nucleotide sequences with the NCBI GenBank database, the Basic Local Alignment Search Tool (BLAST®, https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 3 December 2024) was employed. Amplification products from nested and Mn-PCR-RFLP reactions were visualized on a 1.2% agarose gel under ultraviolet light. Subsequently, Mn-PCR-RFLP products were digested with restriction enzymes in a 20 μL volume and separated on a 3.5% agarose gel. The obtained band patterns were compared with data from ToxoDB (https://toxodb.org/toxo/; accessed on 16 December 2024) and two research papers [32,33] to determine the T. gondii genotype (Table 3).

2.3. Statistical Analysis

Statistical analyses were conducted using IBM SPSS Statistics version 29.0 (Armonk, NY, USA) for the following variables: sex (female, male), age (in years), co-infection (e.g., presence of other intestinal parasites such as Giardia duodenalis or Cystoisospora spp.), stool consistency (formed, diarrhetic), and parasite genotype (T. gondii type I or II, H. hammondi). Only statistically significant results (p ≤ 0.05) are reported. Given that most variables were measured on a nominal scale and the age variable did not meet the assumption of normality, non-parametric tests were applied.

3. Results

Between 2020 and 2024, oocysts smaller than 15 μm in diameter were found in 0.14% (85/61,648) of feline stool samples. The 67 (0.11%) samples were tested positive for T. gondii and 18 (0.03%) for H. hammondi (Figure 2 and Figure 3). No co-infection of T. gondii and H. hammondi was observed. The annual detection rates of T. gondii and H. hammondi are presented in Table 4. Among 67 T. gondii DNA isolates analyzed, 13 belong to clonal lineage I and 54 to lineage II (Figure 4, Figure 5 and Figure 6). Additional information on co-infections, stool consistency, sex, age of cats, and T. gondii strain types is provided in Table 5. Nested and Mn-PCR-RFLP enabled the identification of three T. gondii genotypes: ToxoDB#1 genotype (11.9%; 8/67), ToxoDB#3 (68.7%; 46/67), and ToxoDB#10 (19.4%; 13/67). All 13 genetic markers were successfully amplified, which can be explained by the large number of oocysts excreted in the cat feces (Table 3).
The statistical analyses were based on data obtained from 85 domestic cats (45 females and 40 males), aged approximately 2 months to 18 years. The overall mean age was 2.71 years (SD = 4.09); female cats had a mean age of 1.59 years (SD = 2.43), while males averaged 3.97 years (SD = 5.12).
A chi-square goodness-of-fit test revealed no significant difference in T. gondii prevalence between females (n = 36) and males (n = 31), χ2(1) = 0.37, p = 0.541. Among cats under 1 year of age, T. gondii (n = 45) was significantly more prevalent than H. hammondi (n = 12), χ2(1) = 19.11, p < 0.001. This trend was consistent across the full sample: T. gondii (n = 67) occurred significantly more frequently than H. hammondi (n = 18), χ2(1) = 28.25, p < 0.001. A chi-square test for independence indicated a significant association between age group and the occurrence of diarrhea, with younger cats (<1 year) exhibiting diarrhea more frequently than older individuals, χ2(1) = 7.11, p = 0.008, Cramer’s V = 0.317. Cats infected with T. gondii type I (ToxoDB#10) were significantly younger (mean rank = 20.54, n = 13) compared to those infected with type II (ToxoDB#1 and 3), (mean rank = 37.24, n = 54), as revealed by a Mann–Whitney U test, U = 176.00, Z = –2.78, p = 0.005, r = 0.34. The type of parasite was also significantly associated with the presence of diarrhea, which occurred more frequently in cats shedding T. gondii than in those shedding H. hammondi, χ2(1) = 13.08, p < 0.001, Cramer’s V = 0.424. However, there was no significant association between parasite type and the presence of co-infections, χ2(1) = 0.041, p = 0.839 (Table 4). The lack of detailed information regarding the cats’ origin, lifestyle, and diet limits the possibility of providing a more comprehensive context for the findings.

4. Discussion

Cats and other felids are the only mammals in which the sexual phase of the life cycle of T. gondii or H. hammondi occurs [2,34]. Domestic cats living in close proximity to humans are susceptible to T. gondii infection regardless of age, sex, or breed [35]. During the shedding period, cats can excrete over three million oocysts into the environment via feces [15].
However, the shedding of T. gondii oocysts typically precedes the development of specific antibodies, rendering serological tests ineffective for detecting active oocyst excretion [36]. Lappin et al. emphasize that diagnosing toxoplasmosis in cats through coproscopic examination is unreliable, due to both the brief duration of oocyst shedding and the low sensitivity of this diagnostic method [36]. The oocysts of T. gondii and H. hammondi are morphologically very similar, making them difficult to distinguish through routine microscopic examination [37]. However, certain features observable in histopathological analysis and pathophysiological differences following bioassays in mice, such as the infectivity of tachyzoites and bradyzoites or the ability for congenital transmission, are present in T. gondii but absent during H. hammondi infections [3]. Consequently, correct differentiation of T. gondii oocysts from H. hammondi is essential for epidemiological studies and ensuring public health safety. Therefore, molecular biology techniques are employed as they allow for the rapid identification of the protozoan without performing a bioassay [19].
Berger-Schoch et al. detected small oocysts in 2 out of 252 feline fecal samples. Molecular analysis confirmed T. gondii in one sample and H. hammondi in the other. The T. gondii-positive case was an 11-year-old indoor individual with chronic pneumonia [38]. In our study, T. gondii shedding was also detected in an 18-year-old cat, which had been treated with prednisolone 1 mg/kg, twice daily (Prednicortone 20 mg tablets for dogs and cats, Dechra Regulatory B.V., Best, The Netherlands) for intestinal lymphoma. Researchers suggest that oocyst shedding and acute systemic toxoplasmosis in older cats may be associated with compromised immunity resulting from comorbidities, including infectious diseases such as Feline Immunodeficiency Virus (FIV), Feline Leukemia Virus (FeLV), Feline Infectious Peritonitis (FIP), or immunosuppressive therapy [39,40,41,42,43,44].
Diarrhea is a frequently reported symptom of T. gondii and other coccidian intestinal parasitosis [45,46]. Diarrhea was also reported in 2 out of 60 cats in Latvia and 6 out of 903 cats in Germany, shedding T. gondii oocyst [47,48]. In our research, diarrhea was observed in 80.6% (54/67) of cats shedding T. gondii and 33.3% (6/18) of those shedding H. hammondi.
T. gondii oocysts are rarely diagnosed via coproscopy due to the short duration of shedding. In Poland, Wąsiatycz identified T. gondii oocysts in 0.67% (1/149) of cat stool samples in Poznań [49]. Similarly, in Germany, coproscopic examination conducted between 2004 and 2006 in a private laboratory revealed a comparably low shedding rate (0.1%; 22/20,317). Additionally, only a few individual cats shedding oocysts were identified in studies from France, Austria, and Switzerland: 2/858, 1/994, and 1/5, respectively. In contrast, no oocyst-positive fecal samples were found in the feline population from the Netherlands (n = 966), Denmark (n = 437), or Italy (n = 257) [50]. In Brazil, T. gondii oocyst shedding was observed in 4 out of 237 stray animals (two kittens and two adult cats). Notably, these animals did not have detectable specific IgG antibodies, which suggests a primary infection [51].
The genetic diversity of T. gondii circulating in Europe and North America includes three major clonal lineages: type I, II, and III, which are capable of infecting humans and animals and are also commonly found in the environment (soil and water) [52,53,54,55,56,57]. Although these strains do not differ morphologically, their virulence varies significantly in experimental murine models [55]. Genotyping conducted by Lehmann et al. [58] identified more virulent atypical strains and hybrid isolates of T. gondii, which are significantly more prevalent in South America than in Europe or North America.
In the current study, 67 T. gondii DNA isolates from feline feces were genotyped using nested and multilocus PCR-RFLP. Thirteen of these isolates were identified as type I (ToxoDB#10). The genetic structure of T. gondii strains in cats has been analyzed across various regions globally. In Southern Thailand, among eight T. gondii isolates from cat feces, two were atypical, two were recombinant, one belonged to type I, two to type III, and one to either type II or III [59]. In Iran (Mashhad region), T. gondii type II genotype was identified in the feces of 8 out of 175 stray cats. Additionally, T. gondii DNA was detected in brain tissue and in the feces of 2 out of 31 deceased stray cats [60].
Numerous European studies utilizing multilocus PCR-RFLP have shown that type II is the predominant clonal lineage identified in domestic cats [38,61,62]. However, type I isolates have also been particularly reported in Spain and Italy [63,64]. In our study, type I (T. gondii clonal lineage I) was detected in 13 out of 67 feline fecal samples. For the first time, atypical T. gondii genotypes, distinct from clonal lineages I, II, and III, were identified in Germany in feces from naturally infected cats [65]. Molecular analysis of T. gondii strains responsible for systemic toxoplasmosis in cats also suggests genotype 3 (type II) [66]. The same genotype has also been documented in Germany [65]. In the present study, ToxoDB#3 was detected in 46 out of 67, indicating it as the dominant genotype infecting cats in Central Europe [62].
The high prevalence of this genotype may be related to its widespread occurrence in wild rodents and insectivores, which act as intermediate or paratenic hosts in the life cycle of T. gondii [67]. In Poland, T. gondii DNA, primarily genotype II and III, was detected in tissue samples from 10 wild rodents and insectivores captured in the Lublin Province [68]. In the Mazury Lake District (northeastern Poland), Grzybek et al. reported a T. gondii seroprevalence of 5.5% (32/577) among four rodent species [69]. However, a separate study failed to detect T. gondii DNA in the tissues of seropositive animals [70].
Consumption of raw or undercooked meat is considered one of the primary risk factors for T. gondii infection in domestic cats [71,72,73]. Genetic analysis of T. gondii strains isolated from sheep in Italy revealed infection with the type II clonal lineage [74]. In 19 out of 57 pork meat samples from grocery stores in the United Kingdom, T. gondii DNA of types II and I was detected [75]. In studies conducted in Poland by Sroka et al., T. gondii DNA was detected in 5.4% (175/3223) of meat product samples retailed in Poland. Genetic multilocus analysis was possible for only 61 PCR-positive samples amplified for the B1 gene. Finally, type I, type II, and type III T. gondii lineages were identified in 10 (10.2%), 17 (17.3%), and 48 (49.0%) samples, respectively [76]. These findings suggest that the same genotypes circulating among livestock are likely responsible for infections in domestic cats, particularly those with access to raw or undercooked meat.

5. Conclusions

Based on our study, the detection rate of T. gondii oocysts in feline feces with coproscopy methods was below 1%, and distinguishing them with H. hammondi oocyst without the use of molecular techniques is not feasible. The results also demonstrated considerable genetic diversity of T. gondii among cats across Poland. To the best of our knowledge, this is the first study confirming the presence of type I clonal lineage (ToxoDB#10) in naturally infected cats in Poland. This finding underscores the importance of implementing advanced diagnostics tools for toxoplasmosis, not only in definitive hosts, but also in susceptible species, including humans. The observed prevalence of T. gondii type I raises concerns regarding its ecological impact and potential risks to both human and animal health, highlighting the urgent need for further research into environmental reservoirs, transmission pathways, and the epidemiological significance of different genotypes.

Author Contributions

Conceptualization, D.J.; methodology, D.J.; formal analysis, R.S.; resources, D.J., A.K.M. and J.O.; data curation, D.J., A.K.M. and J.O.; writing original draft preparation, D.J.; writing review and editing, D.J., O.S.-J. and R.S.; visualization, D.J. and R.S.; supervision, O.S.-J., R.S. and D.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was carried out in accordance with the standards recommended by the EU Directive 2010/63/EU for animal experiments and Good Laboratory Practice and the Act of Polish Parliament of 15 January 2015 on protection of animals used for scientific purposes (Journal of Laws 2015, item 266). All procedures were in line with Polish law regulations. Samples were submitted by veterinarians as a part of the laboratory service tests; therefore, the Ethic Commission Agreement was not required.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author(s).

Conflicts of Interest

The authors declare no conflicts of interest.

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Pathogens 14 00444 g001
Figure 1. Oocyst (black arrows) morphologically resembling Toxoplasma gondii or Hammondia hammondi in a feline fecal sample detected by zinc sulfate flotation. Magnification 400×.
Figure 1. Oocyst (black arrows) morphologically resembling Toxoplasma gondii or Hammondia hammondi in a feline fecal sample detected by zinc sulfate flotation. Magnification 400×.
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Pathogens 14 00444 g002
Figure 2. Electrophoresis results of the PCR products on a 2% agarose gel. The amplified PCR product 530 bp T. gondii B1 gene fragment. Lane 1—positive control RH strain of T. gondii, lane 2—negative control (ddH2O), M—marker (Marker 3, A&A Biotechnology, Gdańsk, Poland), lanes 3, 10, 12–13 negative samples, lanes 4–9 and 11 positive samples.
Figure 2. Electrophoresis results of the PCR products on a 2% agarose gel. The amplified PCR product 530 bp T. gondii B1 gene fragment. Lane 1—positive control RH strain of T. gondii, lane 2—negative control (ddH2O), M—marker (Marker 3, A&A Biotechnology, Gdańsk, Poland), lanes 3, 10, 12–13 negative samples, lanes 4–9 and 11 positive samples.
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Pathogens 14 00444 g003
Figure 3. Electrophoresis results of the PCR products on a 2% agarose gel. The amplified PCR product ~240 bp of H. hammondi (lane 4—positive sample). Lanes 1–3 and 5–6 samples were negative for H. hammondi. M—marker (Marker 3, A&A Biotechnology, Gdańsk, Poland).
Figure 3. Electrophoresis results of the PCR products on a 2% agarose gel. The amplified PCR product ~240 bp of H. hammondi (lane 4—positive sample). Lanes 1–3 and 5–6 samples were negative for H. hammondi. M—marker (Marker 3, A&A Biotechnology, Gdańsk, Poland).
Pathogens 14 00444 g003
Pathogens 14 00444 g004
Figure 4. Electrophoresis results of the RFLP-PCR products of T. gondii B1 gene fragment digested with PmlI (Eco72) endonuclease. Lane 1 T. gondii RH strain (type I), lanes 2–8 T. gondii DNA isolates from feline feces; lanes 2–3, 5, 7 (type I), lanes 4, 6, and 8 (type II/III). M—marker (Marker 3, A&A Biotechnology, Gdańsk, Poland).
Figure 4. Electrophoresis results of the RFLP-PCR products of T. gondii B1 gene fragment digested with PmlI (Eco72) endonuclease. Lane 1 T. gondii RH strain (type I), lanes 2–8 T. gondii DNA isolates from feline feces; lanes 2–3, 5, 7 (type I), lanes 4, 6, and 8 (type II/III). M—marker (Marker 3, A&A Biotechnology, Gdańsk, Poland).
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Pathogens 14 00444 g005
Figure 5. Electrophoresis results of the RFLP-PCR products of T. gondii SAG2-3′ digested with HhaI endonuclease. Lane 1 T. gondii RH strain (type I), lanes 2–5 T. gondii DNA isolates from feline feces (type II/III). M—marker (Marker 3, A&A Biotechnology, Gdańsk, Poland).
Figure 5. Electrophoresis results of the RFLP-PCR products of T. gondii SAG2-3′ digested with HhaI endonuclease. Lane 1 T. gondii RH strain (type I), lanes 2–5 T. gondii DNA isolates from feline feces (type II/III). M—marker (Marker 3, A&A Biotechnology, Gdańsk, Poland).
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Pathogens 14 00444 g006
Figure 6. Electrophoresis results of the RFLP-PCR products of T. gondii GRA6 digested with MseI endonuclease. Lane 1 T. gondii RH strain (type I), lanes 2–8 T. gondii DNA isolates from feline feces (type II). M—marker (Marker 3, A&A Biotechnology, Gdańsk, Poland).
Figure 6. Electrophoresis results of the RFLP-PCR products of T. gondii GRA6 digested with MseI endonuclease. Lane 1 T. gondii RH strain (type I), lanes 2–8 T. gondii DNA isolates from feline feces (type II). M—marker (Marker 3, A&A Biotechnology, Gdańsk, Poland).
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Table 1. List of primers used in the screening test to differentiate Hammondia hammondi and Toxoplasma gondii oocysts from feline stool samples.
Table 1. List of primers used in the screening test to differentiate Hammondia hammondi and Toxoplasma gondii oocysts from feline stool samples.
Parasite SpeciesForward PrimerSequence (5′-3′)Reverse PrimerSequence (5′-3′)
Hammondia hammondiHham34FATCCCATTCCGGCTTCAGTCTTTCHham3RACAGCGGAGCCGAAGTTGGTTT
Toxoplasma gondiiTOX4CGCTGCAGGGAGGAAGACGAAAGTTGTOX5CGCTGCAGACACAGTGCATCTGGATT
Table 2. List of primers and corresponding restriction enzymes used in molecular characterization of Toxoplasma gondii strains obtained from feline stool samples.
Table 2. List of primers and corresponding restriction enzymes used in molecular characterization of Toxoplasma gondii strains obtained from feline stool samples.
MarkersPCR External Primers (Sequence 5′-3′)Nested PCR Internal Primers (Sequence 5′-3′)Nested PCR ProductRestriction Enzymes
B1F:TGTTCTGTCCTATCGCAACGF:TCTTCCCAGACGTGGATTTC530 bpPml1
R:ACGGATGCAGTTCCTTTCTGR:CTCGACAATACGCTGCTTGA
SAG1F:GTTCTAACCACGCACCCTGAGF:CAATGTGCACCTGTAGGAAGC390 bpSau96I and HaeII (double digest)
R:AAGAGTGGGAGGCTCTGTGAR:GTGGTTCTCCGTCGGTGTGAG
5′-SAG2F:GCTACCTCGAACAGGAACACF:GCATCAACAGTCTTCGTTGC242 bpSau3AI
R:GAAATGTTTCAGGTTGCTGCR:GCAAGAGCGAACTTGAACAC
3′-SAG2F:TCTGTTCTCCGAAGTGACTCCF:ATTCTCATGCCTCCGCTTC222 bpHhaI
R:TCAAAGCGTGCATTATCGCR:AACGTTTCACGAAGGCACAC
alt. SAG2F:GGAACGCGAACAATGAGTTTF:ACCCATCTGCGAAGAAAACG546 bpHinfI and TaqI (separate digest)
R:GCACTGTTGTCCAGGGTTTTR:ATTTCGACCAGCGGGAGCAC
SAG3F:CAACTCTCACCATTCCACCCF:TCTTGTCGGGTGTTCACTCA225 bpNciI
R:GCGCGTTGTTAGACAAGACAR:CACAAGGAGACCGAGAAGGA
BTUBF:TCCAAAATGAGAGAAATCGTF:GAGGTCATCTCGGACGAACA411 bpBsiE and TaqI (double digest)
R:AAATTGAAATGACGGAAGAAR:TTGTAGGAACACCCGGACGC
GRA6F:ATTTGTGTTTCCGAGCAGGTF:TTTCCGAGCAGGTGACCT344 bpMseI
R:GCACCTTCGCTTGTGGTTR:TCGCCGAAGAGTTGACATAG
C22-8F:TGATGCATCCATGCGTTTATF:TCTCTCTACGTGGACGCC521 bpBsmAI and MboII (double digest)
R:CCTCCACTTCTTCGGTCTCAR:AGGTGCTTGGATATTCGC
C29-2F:ACCCACTGAGCGAAAAGAAAF:AGTTCTGCAGAGTGTCGC446 bpHpyCH4IV and RsaI (double digest)
R:AGGGTCTCTTGCGCATACATR:TGTCTAGGAAAGAGGCGC
L358F:TCTCTCGACTTCGCCTCTTCF:AGGAGGCGTAGCGCAAGT419 bpHaeIII and NlaIII (double digest)
R:GCAATTTCCTCGAAGACAGGR:CCCTCTGGCTGCAGTGCT
PK1F:GAAAGCTGTCCACCCTGAAAF:CGCAAAGGGAGACAATCAGT903 bpAvaI and RsaI (double digest)
R:AGAAAGCTCCGTGCAGTGATR:TCATCGCTGAATCTCATTGC
ApicoF:TGGTTTTAACCCTAGATTGTGGF:GCAAATTCTTGAATTCTCAGTT640 bpAflII and DdeI (double digest)
F:AAACGGAATTAATGAGATTTGAAR:GGGATTCGAACCCTTGATA
Table 3. Toxoplasma gondii genotype number based on the RFLP patterns presented in ToxoDB and referenced from Brennan et al., 2016 [32] and Chen et al., 2025 [33].
Table 3. Toxoplasma gondii genotype number based on the RFLP patterns presented in ToxoDB and referenced from Brennan et al., 2016 [32] and Chen et al., 2025 [33].
Toxoplasma gondiiB1SAG1SAG2 5′+3′SAG2 alt.SAG3BTUBGRA6C22-8C29-2L358PK1ApicoToxoDB
Type I (GT1)IIIIIIIIIIII#10
Type II (ME49)II/
III		II/
III		IIIIIIIIIIIIIIIIIIII#1
Type III (CTG)II/
III		II/
III		IIIIIIIIIIIIIIIIIIIIIIIIIIIIII#2
Cats 5, 7, 8II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Genotypes of Toxoplasma gondii detected in cats feces in Poland 2020–2024
T. gondii DNA isolateSexAgeYearB1SAG1SAG2 5′+3′SAG2 alt.SAG3BTUBGRA6C22-8C29-2L358PK1ApicoToxoDB
Cat 1F5M2020IIIIIIIIIIII#10
Cat 2M7M2020II/
III		II/
III		IIIIIIIIIIIIIIIIIIII#1
Cat 3F4Y2020II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 4F5M2020II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 5M2Y2020II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 6F9M2020II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 7M3M2020II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 8F7M2020II/
III		II/
III		IIIIIIIIIIIIIIIIIIII#1
Cat 9F1Y2020II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 10M18Y2020II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 11F8M2020II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 12F9M2020II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 13M4M2021IIIIIIIIIIII#10
Cat 14M4M2021IIIIIIIIIIII#10
Cat 15M3M2021IIIIIIIIIIII#10
Cat 16F2M2021II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 17M11Y2021II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 18F3Y2021II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 19M10Y2021II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 20F8M2022II/
III		II/
III		IIIIIIIIIIIIIIIIIIII#1
Cat 21M13Y2022II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 22F1Y2022II/
III		II/
III		IIIIIIIIIIIIIIIIIIII#1
Cat 23F6M2022II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 24M8Y2022II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 25M9Y2022II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 26F1Y2022II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 27M1Y2022II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 28M2Y2022II/
III		II/
III		IIIIIIIIIIIIIIIIIIII#1
Cat 29M4Y2023IIIIIIIIIIII#10
Cat 30F7M2023IIIIIIIIIIII#10
Cat 31F1Y2023IIIIIIIIIIII#10
Cat 32F6M2023IIIIIIIIIIII#10
Cat 33M13Y2023II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 34M2Y2023II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 35F4Y2023II/
III		II/
III		IIIIIIIIIIIIIIIIIIII#1
Cat 36M8M2023II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 37F3Y2023II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 38F6Y2023II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 39F4M2023II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 40M2M2023II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 41M3M2023II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 42M16Y2023II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 43M11Y2023II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 44F2M2023II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 45F5M2023II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 46M9M2023II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 47F5M2023II/
III		II/
III		IIIIIIIIIIIIIIIIIIII#1
Cat 48F6M2024IIIIIIIIIIII#10
Cat 49F3Y2024II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 50M8Y2024II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 51M2Y2024II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 52F3M2024II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 53F9M2024II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 54F2Y2024II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 55M8M2024II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 56M2Y2024II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 57M4M2024IIIIIIIIIIII#10
Cat 58F7M2024II/
III		II/
III		IIIIIIIIIIIIIIIIIIII#1
Cat 59F3M2024II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 60F1Y2024II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 61F2Y2024II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 62M11M2024II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 63F4M2024IIIIIIIIIIII#10
Cat 64F6M2024IIIIIIIIIIII#10
Cat 65F6M2024IIIIIIIIIIII#10
Cat 66M7M2024II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Cat 67M7M2024II/
III		II/
III		IIIIIIIIIIIIIIIIIII#3
Table 4. Annual distribution of feline stool samples testing positive for T. gondii and H. hammondi in Poland.
Table 4. Annual distribution of feline stool samples testing positive for T. gondii and H. hammondi in Poland.
Examined Feline
Stool Samples		Toxoplasma gondii
Positive Samples		Hammondia hammondi
Positive Samples
Yearnn%n%
20201780120.6710.06
2021576070.1260.10
202211,64190.0850.04
202319,121190.1040.02
202423,346200.0920.01
Total:61,648670.11180.03
Table 5. Comparison of T. gondii and H. hammondi positive samples according to host age, sex, stool consistency, co-infections, and identified clonal lineage.
Table 5. Comparison of T. gondii and H. hammondi positive samples according to host age, sex, stool consistency, co-infections, and identified clonal lineage.
T. gondiiH. hammondi
Age
<1 Y		Age
≥1		Age
<1		Age
≥1
Total3730810
Male141754
Female231336
Diarrhea361851
Formed stool11239
co-infection9512
Giardia duodenalis4101
Cystoisospora felis2300
Tritrichomonas foetus1111
Toxocara cati2000
no co-infection282578
T. gondii clonal lineage I112--
T. gondii clonal lineage II2628--
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Jańczak, D.; Stryjek, R.; Maj, A.K.; Olszewski, J.; Szaluś-Jordanow, O. PCR Detection, Genotyping, and Differentiation of Toxoplasma gondii from Hammondia hammondi Excreted in the Feces of Cats in Poland Between 2020 and 2024. Pathogens 2025, 14, 444. https://doi.org/10.3390/pathogens14050444

AMA Style

Jańczak D, Stryjek R, Maj AK, Olszewski J, Szaluś-Jordanow O. PCR Detection, Genotyping, and Differentiation of Toxoplasma gondii from Hammondia hammondi Excreted in the Feces of Cats in Poland Between 2020 and 2024. Pathogens. 2025; 14(5):444. https://doi.org/10.3390/pathogens14050444

Chicago/Turabian Style

Jańczak, Dawid, Rafał Stryjek, Aleksandra Kornelia Maj, Jakub Olszewski, and Olga Szaluś-Jordanow. 2025. "PCR Detection, Genotyping, and Differentiation of Toxoplasma gondii from Hammondia hammondi Excreted in the Feces of Cats in Poland Between 2020 and 2024" Pathogens 14, no. 5: 444. https://doi.org/10.3390/pathogens14050444

APA Style

Jańczak, D., Stryjek, R., Maj, A. K., Olszewski, J., & Szaluś-Jordanow, O. (2025). PCR Detection, Genotyping, and Differentiation of Toxoplasma gondii from Hammondia hammondi Excreted in the Feces of Cats in Poland Between 2020 and 2024. Pathogens, 14(5), 444. https://doi.org/10.3390/pathogens14050444

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