Anti-Toxoplasma gondii antibody positivity in humans and selected ruminants in Urmia, Iran: a cross-sectional study

¹Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

^&Corresponding author
Mousa Tavassoli, Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

Introduction    

T. gondii is an intracellular protozoan parasite that infects humans and many warm-blooded animals. Felids are the definitive hosts and shed environmentally resistant oocysts, while humans and livestock may become infected through contaminated soil, water, food, or tissue cysts in raw or undercooked meat [1-4]. Human infection is often asymptomatic, but it may cause severe disease in immunocompromised individuals and adverse fetal outcomes when primary infection occurs during pregnancy [2,3,5,6]. In livestock, toxoplasmosis is relevant because infection in ruminants may indicate environmental exposure and may have implications for animal health and food safety [7-9]. These features make T. gondii an important parasite for studies that consider human, animal, and environmental health together [10].

Although previous studies have reported T. gondii exposure in humans and domestic animals [8,9,11-14], fewer studies have examined humans and multiple ruminant species within the same local setting while comparing blood and milk samples [7,15,16]. This gap limits understanding of how frequently antibodies are detected across host groups and sample types in shared human-animal environments. Urmia, Iran, includes urban, peri-urban, and livestock-associated settings where humans and domestic ruminants may be sampled within the same geographic area [13]. The rationale for this investigation was therefore to describe anti-T. gondii antibody detection in humans and selected ruminants from this setting using a cross-sectional design.

The primary objective was to estimate anti-T. gondii IgG positivity and composite IgG and/or IgM positivity in blood and milk samples from humans, cows, buffaloes, sheep, and goats in Urmia, Iran. Separate IgM-positive counts were unavailable in the analysed laboratory summary and could not be estimated independently. A secondary objective was to compare antibody-positive proportions across host groups and sample types.

Methods     

Study design: this cross-sectional study estimated anti-T. gondii IgG positivity and composite IgG and/or IgM positivity in human and selected ruminant blood and milk samples from Urmia, Iran. Separate IgM-positive counts and sample-level IgG/IgM combinations were unavailable, so IgM positivity could not be estimated independently. Exposure and antibody status were assessed at sample collection; no follow-up was performed.

Setting and dates: this cross-sectional study was conducted in Urmia, West Azerbaijan Province, Iran, from June 2025 to June 2026. Human samples were collected from Imam Khomeini Hospital. Animal samples were collected from dairy farms and smallholder livestock farms in Urmia. Laboratory testing was performed at the Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran, from June 2025 to June 2026.

Sample collection

Animal participants and eligibility: the animal study population comprised cows, buffaloes, sheep, and goats sampled from dairy and smallholder livestock farms in Urmia, Iran. Animals were selected by convenience sampling during the study period, and milk was collected only from lactating females. For each species, Table 1 reports 50 blood specimens and 50 milk specimens. Available records do not confirm whether these specimens were paired within the same 50 animals. Animal identifiers or sampling logs should therefore be checked before submission. Unless pairing is verified, blood and milk results should be treated as separate specimen groups, without within-animal comparison. Animals were excluded when sampling was clinically unsuitable or owner permission was not obtained (Table 1).

Human participants and eligibility: human samples were obtained at Imam Khomeini Hospital from women providing blood specimens (n=50), children providing blood specimens (n=50), and lactating women providing milk specimens (n=50) between June 2025 and June 2026. Available records do not confirm whether the 50 milk providers were distinct from, or overlapped with, the 50 women who provided blood. Participant identifiers or sampling logs should therefore be checked before submission. Unless overlap is verified, the three denominators should be treated as separately reported specimen groups and should not be summed to infer 150 unique participants. Participants were selected by convenience sampling when specimen collection was clinically and ethically permissible. Children were included only with permission from a parent or legal guardian. Individuals were excluded when consent or guardian permission was unavailable or specimen collection was clinically unsuitable.

Variables: the primary outcome variable was anti-T. gondii antibody positivity in serum or milk. "IgG and/or IgM positive" replaces the ambiguous term "combined antibody result" and is defined as a sample reactive for at least one antibody class (IgG-positive, IgM-positive, or positive for both IgG and IgM). This composite outcome is not the arithmetic sum of the separate IgG and IgM counts; each sample contributes once. Because the analysed laboratory summary did not include a separate IgM-positive count or sample-level IgG/IgM combinations, independent IgM prevalence and the numbers positive for IgM alone or for both antibodies could not be determined. Sample type was classified as blood or milk. Host group was classified as human, cow, buffalo, sheep, or goat; human subgroups were reported as women, children, and milk-sample providers. No additional measured exposure, predictor, confounder, or effect-modifier variables were available for analysis.

Sample preparation

Blood processing: blood samples were centrifuged at 3,000 rpm for 15 minutes to separate serum. Serum aliquots were stored at -20°C until serological testing. All samples were labelled using a unique code to maintain traceability and reduce transcription errors.

Milk processing: milk samples were centrifuged at 2,500 rpm for 10 minutes to remove the fat layer. The aqueous fraction was separated and stored at -80°C until serological testing.

Data sources and measurement: data sources were laboratory test results from processed serum and milk samples and recorded host and sample-type information. All reported groups were assessed using the same serological approach. ELISA-based antibody detection is commonly used for epidemiological assessment of T. gondii exposure, although performance may vary by sample type, antigen, antibody target, and assay format [17,18]. The reviewed manuscript and available laboratory records did not specify the ELISA kit manufacturer, catalogue number, sample dilution, incubation time and temperature, plate-reading wavelength, exact cut-off formula, or complete quality-control criteria. These details are important for reproducibility and should be added if they can be verified from the original kit insert, laboratory worksheets, instrument records, or procurement documents. No assay parameter was inferred from external studies.

Bias: potential sources of bias include selection bias related to convenience sampling, information bias from limited participant or animal-level data, and misclassification bias related to serological interpretation. To reduce measurement variation, serum and milk samples were processed using stated laboratory procedures and tested using the same serological approach. Samples were labelled using unique codes to maintain traceability and reduce transcription errors.

Study size: the study included 50 samples in each reported human and animal subgroup. No formal sample-size calculation was available in the current manuscript file; therefore, the analysed sample size was treated as feasibility-based convenience sampling.

Quantitative variables: the main quantitative variables were the number tested, number positive, and percentage positive for each host group and sample type. No continuous quantitative variables or grouped risk-factor variables are reported in the current manuscript.

Ethical considerations: the study was conducted in accordance with the Declaration of Helsinki and relevant ethical principles and guidelines. The Research Ethics Committee of Urmia University of Medical Sciences, Urmia, Iran, approved the study under approval number IR-UU-AEC-3.85.

Serological testing: 1) Serum and milk samples were tested for anti-T. gondii immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies using enzyme-linked immunosorbent assay (ELISA). The available laboratory summary retained IgG-specific results and a composite result indicating reactivity for IgG and/or IgM. It did not retain separate IgM-positive counts or the IgG/IgM overlap needed to derive them; therefore, IgM positivity could not be reported independently; 2) ELISA testing was performed for anti-T. gondii antibody detection in processed serum and milk samples according to the laboratory protocol used at the Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University. Positive, negative, and blank controls were included during testing. Results were interpreted according to the assay cut-off used by the laboratory. For reproducibility, the final manuscript should report the kit manufacturer and country, catalogue or product number, antibody target and assay format, serum and milk dilutions, reagent volumes, incubation times and temperatures, washing procedure, substrate and stop-solution conditions, plate-reading wavelength, control acceptance criteria, and the precise formula used to classify samples as positive, negative, or equivocal. Because these details could not be verified from the records reviewed, they are not reported here and should be inserted only after confirmation from the original laboratory documentation; 3) IgG reactivity was interpreted as evidence of previous exposure, whereas IgM reactivity was considered suggestive of possible recent exposure. Because isolated or persistent IgM reactivity can be difficult to interpret, IgM positivity was not treated as definitive evidence of acute infection without confirmatory testing such as repeat serology, IgG avidity testing, or molecular confirmation [2,18].

Statistical analysis: data were entered into Microsoft Excel and analysed descriptively. Frequencies and percentages were calculated for each host group and sample type. Antibody positivity was calculated as the number of positive specimens divided by the number tested. The term "seroprevalence" is reserved for serum-based estimates; results that include milk specimens are described as antibody positivity or antibody detection. Two-sided 95% confidence intervals (CIs) for each binomial proportion were calculated using the Wilson score method, which provides more informative interval estimates than the normal approximation when sample sizes or positive counts are small. Because the study objective was descriptive and individual-level exposure variables were unavailable, no adjusted, subgroup, interaction, or sensitivity analyses were performed. The laboratory summary indicated no missing outcome data for the reported positive-result counts.

Results     

Analysed samples: a total of 50 specimens were analysed for each reported human and animal subgroup. The reported human specimen groups comprised women's blood (n=50), children's blood (n=50), and human milk (n=50); blood and milk specimens from cows, buffaloes, sheep, and goats were also analysed. The available records did not establish whether the human milk specimens came from the same women represented in the women's blood group; therefore, these denominators describe specimen groups rather than confirmed unique participants. For each animal species, the reported denominator was 50 for blood and 50 for milk; the available records did not establish whether the two specimen sets came from the same 50 animals.

Descriptive data: the available descriptive variables were host group and sample type. No missing laboratory outcome data were indicated for the reported positive-result counts.

Outcome estimates: Table 1 presents the numbers and percentages of specimens positive for anti-T. gondii antibodies by host group and sample type. Each estimate is accompanied by a two-sided 95% Wilson CI. The table reports IgG-positive results and, where available, composite IgG and/or IgM-positive results. A separate IgM column is not presented because the laboratory summary did not provide separate IgM-positive counts or sample-level IgG/IgM combinations from which IgM-only or dual-positive results could be derived.

Anti-T. gondii antibody positivity in human blood and milk samples: among women's blood specimens, 7/50 (14.0%) were IgG and/or IgM positive and 4/50 (8.0%) were IgG positive. Among children's blood specimens, 2/50 (4.0%) were IgG positive. Among human milk specimens, 3/50 (6.0%) were IgG and/or IgM positive.

Among ruminant blood specimens, IgG and/or IgM positivity was 9/50 (18.0%) in cows, 1/50 (2.0%) in buffaloes, 8/50 (16.0%) in sheep, and 11/50 (22.0%) in goats. IgG seroprevalence was 6/50 (12.0%) in cows, 1/50 (2.0%) in buffaloes, 5/50 (10.0%) in sheep, and 6/50 (12.0%) in goats. Among ruminant milk specimens, IgG antibody positivity was 4/50 (8.0%) in cows, 1/50 (2.0%) in buffaloes, 3/50 (6.0%) in sheep, and 4/50 (8.0%) in goats.

Discussion     

Principal findings and comparison with other studies: this cross-sectional study detected anti-T. gondii antibodies in humans and all selected ruminant species sampled in Urmia, Iran. In ruminant blood, composite IgG and/or IgM positivity was highest in goats at 11/50 (22.0%), followed by cows at 9/50 (18.0%), sheep at 8/50 (16.0%), and buffaloes at 1/50 (2.0%). IgG seroprevalence was 12.0% in cows, 2.0% in buffaloes, 10.0% in sheep, and 12.0% in goats. These estimates should be interpreted in light of substantial geographic and methodological heterogeneity in Iranian and international seroprevalence studies [12,13,19-22]. In milk, IgG antibody positivity was 8.0% in cows, 2.0% in buffaloes, 6.0% in sheep, and 8.0% in goats. Previous milk-based studies have also reported antibody detection in ruminant milk; however, milk antibody positivity does not establish the presence of viable parasites or milk-borne transmission [7,15,16,22].

Interpretation: the findings indicate exposure to T. gondii among the sampled humans and ruminants. Interpretation should remain cautious because the study was descriptive, relied on unadjusted proportions, and lacked risk-factor data, confounder adjustment, subgroup modelling, and sensitivity analyses. The results are broadly consistent with reports of T. gondii exposure in humans and domestic animals [1,2,8,11-14,19]. Differences among host groups should not be interpreted causally or as evidence of specific transmission pathways because relevant exposure data were unavailable. Antibody positivity in milk indicates antibody reactivity in the tested specimens, not infectious milk [15,16,20].

Generalisability and future research: the results are most applicable to the sampled humans and ruminants from the study setting in Urmia, Iran. Generalisability to all people, livestock, farms, or regions in Iran is limited because the sampling frame, recruitment sites, and selection method are not fully described and because each subgroup included a limited number of samples [23,24]. Future studies should use clearly defined sampling frames, larger sample sizes, documented exposure variables, and confirmatory molecular testing to improve external validity and clarify possible routes of exposure.

Limitations: this study has limitations related to design, precision, measurement, and potential bias. The cross-sectional design estimates antibody positivity at one point in time and cannot establish when infection occurred or the direction of transmission. The limited subgroup size may reduce precision and may make the reported percentages unstable, especially for groups with few positive samples.

The 95% confidence intervals presented in Table 1 are accordingly wide for several estimates, particularly those based on one to three positive specimens, and should be considered when comparing host groups or sample types. If participants and animals were selected according to availability, selection bias may have occurred; this could overestimate or underestimate antibody positivity depending on whether sampled individuals or animals had higher or lower exposure than the wider population. Incomplete information on age, sex, pregnancy status, animal contact, cat exposure, food habits, raw milk consumption, and animal-management practices prevents assessment of confounding and limits interpretation of observed differences between groups. ELISA detects antibodies and does not confirm viable parasites, active infection, or infectivity of milk samples, which may overstate the implication of milk results if interpreted as evidence of transmission. Separate IgM-positive counts and sample-level IgG/IgM combinations were unavailable; therefore, IgM positivity could not be estimated independently, and the composite IgG and/or IgM result cannot distinguish IgM-only from dual-positive samples.

The available records also did not confirm whether the 50 blood and 50 milk specimens reported for each animal species were paired within the same animals. Consequently, the blood and milk findings should be interpreted as separate specimen-level estimates unless animal-level sampling records verify pairing. The absence of verified information on the ELISA manufacturer, catalogue number, sample dilution, incubation conditions, reading wavelength, cut-off calculation, and complete quality-control criteria also limits reproducibility and prevents independent evaluation of assay comparability across host species and sample types. If these details can be recovered from the original laboratory records, they should be incorporated before submission; otherwise, this limitation should remain explicit. These limitations mean that the findings should be interpreted as evidence of antibody detection in the tested samples rather than proof of transmission route or population-level prevalence.

Conclusion     

This study detected anti-T. gondii antibodies in human and ruminant specimens from Urmia, Iran. Among the animal species tested, goats had the highest blood-sample antibody positivity and buffaloes the lowest. IgG antibody positivity in milk indicates antibody reactivity but does not confirm viable parasites or milk-borne transmission. Larger studies with defined sampling frames, detailed exposure data, complete assay documentation, and molecular confirmation are needed to characterize infection patterns in the study area.

Competing interests     

The authors declare no competing interests.

Authors' contributions     

All authors contributed to the conception and design of the study, data interpretation, manuscript revision, and approval of the final version; Fatimah Mohsin Mousa contributed to sample collection, laboratory work, data organization, and drafting of the manuscript; Mousa Tavassoli contributed to supervision, methodology, laboratory oversight, and critical revision of the manuscript; Mohammad Yakhchali contributed to supervision, interpretation of findings, and critical revision of the manuscript. All the authors read and approved the final version of this manuscript.

Acknowledgments     

The authors acknowledge the staff of Imam Khomeini Hospital and the Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, for their assistance with sample collection, laboratory processing, and institutional support. The authors also thank the participating individuals, livestock owners, and animal handlers for their cooperation during the study.

Table     

Table 1: anti-T. gondii antibody positivity in blood and milk samples from humans and selected ruminants

References