Beyond the Primer: Anatomical Site and Cyst Viability Drive the Molecular Detection of Taenia saginata in Slaughterhouse Samples

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Jéssica, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9283419/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 15 You are reading this latest preprint version Abstract Background Bovine cysticercosis, caused by the larval stage of Taenia saginata , results in significant economic losses for the beef industry due to carcass condemnation. While routine post-mortem inspection is mandatory, visual diagnosis lacks specificity, particularly for degenerated or calcified cysts. This study evaluated the diagnostic performance of three conventional PCR assays—a widely used LSU rRNA target (328 bp), a previously described COX1 target (253 bp), and a novel COX1 target (428 bp) designed herein—as complementary tools to visual inspection. A total of 232 suspect macroscopic lesions (180 calcified cysts, 12 viable cysts, and 40 non-specific lesions) were collected from cattle slaughtered in Mato Grosso do Sul, Brazil. Results Our results demonstrated no statistically significant difference in diagnostic sensitivity among the three primer sets ( p > 0.05), indicating that molecular confirmation is not limited by primer selection. Instead, molecular detection was profoundly driven by cyst viability and anatomical location ( p < 0.001). While viable cysts yielded high positivity rates (up to 66.7%), detection in calcified lesions dropped to approximately 28.9% due to severe DNA degradation. Crucially, calcified hepatic lesions exhibited drastically lower PCR positivity (11.4%–15.2%) compared to those in the heart (46.0%–54.0%) and head muscles (50.0%–60.0%). Conclusions These findings indicate that visual inspection of hepatic tissue significantly overestimates the prevalence of T. saginata , likely due to the misclassification of non-parasitic granulomas. To improve diagnostic accuracy and prevent unwarranted carcass condemnations, molecular confirmation efforts and routine sanitary evaluations should prioritize suspect lesions in the heart and striated muscles over those found in the liver. Bovine cysticercosis Taenia saginata PCR Molecular diagnosis Meat inspection Food safety Figures Figure 1 Background Brazil plays a prominent role in the global beef market, consolidating its position as the world’s largest beef exporter (ABIEC, 2025). In this context, strict hygienic–sanitary control throughout the production chain is essential to ensure food safety and maintain access to international markets. Among parasitic diseases affecting both public health and the meat industry, bovine cysticercosis—caused by the larval stage of the zoonotic cestode Taenia saginata —stands out as a major cause of partial or total carcass condemnation, leading to significant economic losses (Jorga et al., 2025 ). The taeniasis–cysticercosis complex represents a critical public health concern, directly involving the human–animal interface and environmental contamination through the shedding of infective eggs (Eichenberger et al., 2020 ; Elbarbary et al., 2025 ). In Brazil, according to the Regulation of Industrial and Sanitary Inspection of Products of Animal Origin (RIISPOA) (Brasil, 2017 ), the diagnosis of bovine cysticercosis relies on post-mortem macroscopic identification, classifying cysticerci as viable or calcified. Although routine meat inspection is essential, visual examination presents inherent limitations. Degenerated or calcified cysticerci are notoriously difficult to distinguish from other pathological lesions. Consequently, misclassification frequently occurs, resulting in the overestimation of infection rates and the inappropriate condemnation of carcasses (El-Sayad et al., 2021 ). Recent studies reinforce these diagnostic challenges. In the state of Tocantins, Brazil, visual inspection identified a low frequency of cystic lesions, yet molecular analysis and histopathology confirmed T. saginata in only a fraction of these samples, identifying many as non-specific granulomatous lesions or hydatid disease (Figueiredo et al., 2019 ). Similarly, El-Sayad et al. ( 2021 ) demonstrated that visual inspection significantly overestimated the occurrence of T. saginata cysticerci in cattle slaughtered in Egypt when compared to PCR confirmation. Together, these findings highlight the limited specificity of macroscopic diagnosis and emphasize the need for complementary approaches. Molecular techniques, particularly polymerase chain reaction (PCR), are widely utilized for their high analytical sensitivity and specificity (González et al., 2006 ; Cuttell et al., 2013 ; Uys et al., 2023 ). Nevertheless, PCR performance is highly susceptible to field variables, including lesion stage, DNA degradation in mineralized cysts, and primer efficiency (Abuseir et al., 2006 ; Uys et al., 2023 ). We hypothesized that incorporating conventional PCR as a complementary tool would significantly improve the specificity of visual diagnosis, particularly in lesions classified as degenerated or calcified. Furthermore, we hypothesized that differences in primer sequences and the anatomical origin of the lesion could influence diagnostic reliability. Accordingly, this study aimed to evaluate the performance of previously described primer sequences against a novel primer set standardized herein. Assays were conducted using slaughterhouse samples from Mato Grosso do Sul, Brazil, to assess the practical applicability of PCR as a complementary diagnostic tool in routine meat inspection. Methods Study design and sample collection This study was conducted between April 2023 and June 2024 in the state of Mato Grosso do Sul, Brazil, to evaluate and compare the diagnostic performance of three conventional PCR assays for detecting Taenia saginata DNA in bovine tissue. A total of 232 tissue samples were analyzed. The majority of the samples (n = 224) were collected from cattle slaughtered at an establishment operating under the Federal Inspection Service (SIF), while the remaining samples (n = 8) were sourced from a slaughterhouse under the State Inspection Service (SIE). All samples were collected at the Final Inspection Department from carcasses that had been diverted due to suspected cystic lesions identified during routine post-mortem examination. Tissue fragments were excised from standard anatomical inspection sites, including the heart, liver, diaphragm, masseter and pterygoid muscles, and esophagus. Each sample was individually cataloged (recording carcass number, batch number, anatomical location, and collection date), placed in a sterile plastic container, maintained under refrigeration, and transported to the Animal Health Laboratory at Embrapa Beef Cattle for molecular analysis. To ensure sufficient material for DNA extraction, cystic structures were collected along with a small margin of adjacent tissue. Macroscopic classification Macroscopic classification and subsequent carcass disposition strictly adhered to the criteria established by Brazilian sanitary inspection regulations, which define infection severity based on the number and anatomical distribution of the cysts. Based on slaughterhouse records and visual inspection, the 232 collected samples were divided into three distinct categories: calcified cysts (n = 180, which included all eight samples from the state-inspected slaughterhouse), viable cysts (n = 12), and visually negative or inconclusive lesions (n = 40). This last group served as specificity controls and comprised non-specific macroscopic findings, such as hepatic fatty nodules, necrotic or inflammatory liver nodules, uncharacterized calcified liver nodules, unspecified cardiac nodules, focal connective tissue in the liver and heart, and hepatic sarcocystosis lesions. DNA extraction and quality assessment Genomic DNA was extracted from approximately 25 mg of homogenized tissue using the PureLink Genomic DNA Mini Kit (Invitrogen, USA), according to the manufacturer’s instructions. DNA concentration and purity were determined using a NanoDrop 2000c spectrophotometer (Thermo Scientific, USA). DNA integrity was verified by electrophoresis on a 0.8% agarose gel. PCR assays Three conventional PCR assays for the detection of T. saginata were evaluated. The first assay, designed and standardized in this study, targeted a 428-base pair (bp) fragment of the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene. Specific primers were designed using the Primer-BLAST tool (NCBI): forward (5′-GGATTGCCTCGTCGTGTTTG-3′) and reverse (5′-CTAAGCATGATGCAAAAGGC-3′). Amplification reactions (20 µL) contained GoTaq Colorless Master Mix 2X (Promega, USA), 0.25 µM of each primer, and 200 ng of template DNA. Thermal cycling was performed in an MJ Mini Personal Thermal Cycler (Bio-Rad, USA), consisting of an initial denaturation at 95°C for 10 min, followed by 35 cycles of 94°C for 1 min, 51°C for 1 min, and 72°C for 40 s, with a final extension step at 72°C for 7 min. The second assay, previously described by Jardim et al. ( 2006 ), targeted a 328-bp fragment of the large subunit (LSU) rRNA gene using the primers TBR-3 (generic forward, 5’-GGCTTGTTTGAATGGTTTGACG-3’) and TBR-4 ( T. saginata -specific reverse, 5’-CGACTCATGAAGATAAACAAGGT-3’). The PCR reactions (20 µL) were performed using the same reagent concentrations as the first assay. The cycling program consisted of an initial denaturation at 94°C for 2 min, followed by 40 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 1 min, concluding with a final extension at 72°C for 5 min. The third assay, adapted from El-Dakhly et al. ( 2023 ), amplified a 253-bp fragment of the COX1 gene using the forward (5′-GGGTGCTGGTATAGGGTGGACT-3′) and reverse (5′-ACGTAAATAAATAAGCCCACAATATT-3′) primers. Amplification reactions (25 µL) contained GoTaq Colorless Master Mix 2X, 0.3 µM of each primer, 200 ng of template DNA, and nuclease-free water to reach the final volume. The cycling conditions included an initial denaturation at 94°C for 4 min, followed by 35 cycles of 94°C for 30 s, 59°C for 1 min, and 72°C for 1 min, with a final extension step at 72°C for 7 min. To assess the presence of PCR inhibitors in samples that tested negative for T. saginata , an internal control PCR targeting the bovine DGAT1 gene was performed. This primer pair amplified a 282 bp fragment of this constitutive gene, confirming DNA quality and amplification competence. PCR products from all assays were separated by electrophoresis on 1.5% agarose gels in 1× TBE buffer at 100–120 V. A 1 kb Plus DNA ladder (Invitrogen, USA) was used as a molecular size marker. Gels were stained with a commercial nucleic acid dye and visualized under ultraviolet light. Analytical sensitivity assessment The analytical sensitivity of the standardized assay was evaluated using a 1:2 serial dilution of DNA extracted from viable T. saginata cysticerci (initial concentration of 200 ng/µL). The detection limit was defined as the lowest concentration that produced a clear, reproducible band on the 1.5% agarose gel. Statistical analysis To evaluate the detection capability across different molecular targets, the positivity frequencies of three PCR primer sets were analyzed: COX1 (428 bp; designed in this study), LSU rRNA (328 bp), and a second COX1 target (253 bp). To determine if PCR positivity was significantly associated with the macroscopic classification of the lesions (viable cysts, calcified cysts, or other lesions), the Chi-square test or Fisher's Exact test was employed, depending on the expected cell frequencies. Furthermore, because the same 232 samples were evaluated across all three molecular targets (paired data), Cochran's Q test was used to assess whether there was a statistically significant difference in overall diagnostic sensitivity among the three primers. All statistical analyses were performed using R software version 4.3.2 (R Core Team, 2023 ). A significance level of p < 0.05 was adopted for all analyses. Results Evaluation of analytical sensitivity demonstrated the following detection limits: 0.48 ng for the COX1 (428 bp) primer pair, 0.97 ng for LSU rRNA (328 bp), and 1.95 ng for COX1 (253 bp). These results indicate that the novel COX1 (428 bp) assay yielded the highest analytical sensitivity (Fig. 1 ). Molecular detection of T. saginata DNA varied significantly depending on the macroscopic developmental stage of the lesion, confirming the profound impact of cyst degeneration on PCR performance ( p < 0.001). For viable cysts (n = 12), positivity rates were high, reaching 50.0% for COX1 (428 bp), 58.3% for LSU rRNA (328 bp), and 66.7% for COX1 (253 bp). In contrast, detection in calcified cysts (n = 180) dropped substantially to 26.7%, 30.0%, and 28.9%, respectively, reflecting severe DNA degradation in chronic lesions. Samples macroscopically classified as 'other lesions' (n = 40) exhibited baseline positivity rates of only 2.5% to 5.0%, highlighting the high specificity of the assays. When comparing the overall diagnostic performance of the three molecular targets across the 232 paired samples, the LSU rRNA target yielded the highest absolute number of positive detections (27.1%), followed by the 253 bp COX1 target (26.3%) and the 428 bp COX1 target (23.7%). However, Cochran's Q test revealed that these variations were not statistically significant ( p > 0.05), indicating that no single molecular target demonstrated absolute diagnostic superiority over the others in routine field samples (Table 1 ). Table 1 Comparison of PCR positivity across three molecular targets for the detection of Taenia saginata according to macroscopic lesion stage. Sample classification Total (n) COX1 (428 bp) [This study] Pos COX1 (428 bp) [This study] Neg LSU rRNA (328 bp) [Jardim et al.] Pos LSU rRNA (328 bp) [Jardim et al.] Neg COX1 (253 bp) [El-Dakhly et al.] Pos COX1 (253 bp) [El-Dakhly et al.] Neg Calcified cyst 180 48 132 54 126 52 128 Viable cyst 12 6 6 7 5 8 4 Other lesions* 40 1 39 2 38 1 39 Total 232 55 177 63 169 61 171 *Includes macroscopic findings such as various hepatic nodules (fatty, necrotic, inflammatory, calcified, and unspecified), cardiac nodules, connective tissue in the liver and heart, and hepatic sarcocystosis. Crucially, organ-stratified analysis revealed that anatomical location profoundly impacts molecular confirmation rates, particularly for chronic lesions ( p < 0.001). Among calcified cysts, samples from the liver (n = 105) exhibited drastically reduced PCR positivity (11.4% to 15.2%). In sharp contrast, calcified cysts in the heart (n = 50) and head muscles (n = 20) demonstrated substantially higher molecular positivity rates (46.0%–54.0% and 50.0%–60.0%, respectively). For viable cysts, found exclusively in the heart and masseter muscle, detection rates remained consistently high (66.7% to 77.8%). This marked discrepancy suggests that visual inspection of the liver suffers from low specificity (due to misclassification of non-parasitic granulomas) or that the hepatic microenvironment accelerates the degradation of parasite DNA (Table 2 ). Table 2 Comparison of PCR positivity for Taenia saginata across three molecular targets according to organ and macroscopic lesion stage. Organ Cyst Type Total (n) COX1 (428 bp) Pos COX1 (428 bp) Neg LSU rRNA (328 bp) Pos LSU rRNA (328 bp) Neg COX1 (253 bp) Pos COX1 (253 bp) Neg Liver Calcified 105 16 89 13 92 12 93 Heart Calcified 50 24 26 23 27 27 23 Heart Viable 3 2 1 1 2 2 1 Masseter Muscle Calcified 17 8 9 8 9 9 8 Masseter Muscle Viable 9 6 3 5 4 5 4 Pterygoid Muscle Calcified 3 2 1 2 1 3 0 Diaphragm Calcified 1 1 0 0 1 1 0 Mixed Samples* Calcified 4 1 3 2 2 2 2 TOTAL 192 60 132 54 138 61 131 *Mixed samples represent combined adjacent tissues occasionally collected from highly calcified or diffuse areas. Discussion Routine post-mortem inspection of bovine carcasses remains the primary line of defense against the transmission of T. saginata (Scandrett et al., 2012 ). However, the subjective nature of macroscopic evaluation often leads to diagnostic uncertainty, particularly with degenerated or calcified lesions (El-Sayad et al., 2021 ). The present study evaluated the diagnostic performance of three conventional PCR assays, revealing that while molecular tools are highly specific, their sensitivity in the field is profoundly dictated by the developmental stage of the cyst and its anatomical location. Consistent with previous literature (Costa et al., 2012 ), our results demonstrated a significant decline in PCR positivity as cysticerci degenerate. Viable cysts contain intact parasitic cells with highly preserved DNA, making them ideal targets. In contrast, calcification represents the end-stage of a severe host-parasite inflammatory response (Peixoto et al., 2018 ). Macroscopically and histologically, degenerated cysts are characterized by caseous necrosis, severe cellular infiltration, and eventual mineralization, culminating in the loss of identifiable parasitic features (Elbarbary et al., 2025 ). This caseous necrosis physically entraps remaining structures and severely degrades nucleic acids. Consequently, the lower molecular detection rate in calcified lesions represents a biological limitation of the sample rather than a failure of the PCR assays. The high specificity of the assays was corroborated by the minimal positivity (2.5%–5.0%) observed in the "other lesions" category (González et al., 2006 ). Interestingly, no statistically significant difference in diagnostic performance was observed among the three molecular targets evaluated ( p > 0.05). This finding is highly relevant, indicating that the current bottleneck in the molecular confirmation of bovine cysticercosis is the intrinsic biological degradation of the target DNA, not primer design. It also validates the newly designed COX1 (428 bp) primers as a reliable, interchangeable tool for future epidemiological studies. The most striking and novel finding of this study emerged from the organ-stratified analysis. The profound discrepancy in molecular confirmation between calcified cysts in the liver (11.4%–15.2%) and those in the heart and head muscles (46.0%–60.0%) provides a critical biological explanation for the low agreement between visual inspection and PCR. This stark contrast supports two primary hypotheses. First, visual inspection of the liver suffers from inherently low specificity. As a major filtering organ, the liver is susceptible to various pathological insults resulting in granulomatous and calcified lesions (e.g., hepatic fascioliasis, hydatidosis, bacterial abscesses). During the rapid pace of an industrial slaughter line, these non-parasitic granulomas are easily misclassified as calcified C. bovis (Costa et al., 2012 ). These observations align with previous reports analyzing the macroscopic profile of cysticercosis. Peixoto et al. ( 2018 ) highlighted that hepatic cysticerci are predominantly non-viable (71.0% to 87.5%). Furthermore, Costa et al. ( 2012 ) emphasized that non-viable cysticercus lesions are easily confused with other etiologies, reducing the specificity of gross anatomical examination. This explains why high visual occurrence rates of hepatic cysticerci often correspond to low true-positive rates upon histological or molecular confirmation (Peixoto et al., 2018 ). Second, distinct tissue microenvironments likely dictate DNA preservation. The liver is an enzymatically aggressive environment with a robust local immune system (e.g., Kupffer cells). It is highly probable that the clearance of parasitic DNA occurs at a much faster rate in the hepatic parenchyma than in striated muscle tissue, where calcified granulomas may retain remnant DNA for extended periods. These findings have direct economic implications for the global beef industry. Relying solely on macroscopic inspection for hepatic lesions leads to a massive overestimation of bovine cysticercosis, resulting in the unnecessary condemnation or costly cold-treatment of carcasses possessing only non-specific hepatic scars. By demonstrating that the vast majority of PCR-negative "calcified cysts" are clustered in the liver, this study advocates for a rigorous reevaluation of sanitary condemnation criteria. Despite these robust findings, this study has certain limitations that warrant consideration. The relatively low number of viable cysts (n = 12) compared to calcified lesions (n = 180) reflects the natural frequency of chronic infections encountered in slaughterhouse lines, but restricts broader statistical inferences regarding early-stage cysticerci. Additionally, the initial sample screening relied on the routine visual inspection conducted by local abattoir staff. While this accurately reflects real-world industry practices and validates the need for molecular confirmation, it inherently carries the subjectivity of visual triaging. Future studies incorporating randomized carcass sampling regardless of gross macroscopic findings could further elucidate the true baseline prevalence of T. saginata . Conclusions Conventional PCR is a highly specific complementary tool for diagnosing bovine cysticercosis, though its sensitivity is intrinsically limited by the severe DNA degradation characteristic of calcified cysts. Our findings validate the newly designed COX1 (428 bp) primer set as a reliable alternative to previously established targets. Crucially, this study reveals that the anatomical location of a lesion dictates molecular detection rates. The vast majority of diagnostic discrepancies between visual inspection and PCR originate in the liver, strongly suggesting that macroscopic examination of hepatic tissue significantly overestimates the prevalence of T. saginata due to the misclassification of non-parasitic granulomas. To improve epidemiological surveillance and mitigate the economic impact of unwarranted carcass condemnations, we strongly recommend that future molecular confirmation efforts—and routine sanitary evaluations—prioritize suspect lesions identified in the heart and striated muscles over those found in the liver. Declarations Ethics approval and consent to participate No animals were slaughtered specifically for the purposes of this study. All biological samples were obtained from cattle processed for commercial purposes under official sanitary inspection. Sample collection occurred during routine post-mortem inspection procedures at federally and state-inspected abattoirs, in accordance with Brazilian legislation governing the industrial and sanitary inspection of products of animal origin (Decree No. 9,013/2017 and subsequent amendments). Because the study was based exclusively on materials collected during routine inspection and did not involve experimental intervention or live animal handling, approval by an institutional animal ethics committee was not required. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil – Finance Code 001. This research was also funded by the project “Beef Cattle National Science and Technology Institute,” supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Grant No. 408696/2024-9), and by CNPq Grants No. 440559/2022-7 and 315857/2021-8. Author Contribution ***E.L.C.S., L.P.C., J.C.K.M., A.P.F.,*** *and* ***A.C.L.A.*** *conducted the experiments.* ***F.R.A., F.A.B., P.H.D.C.,*** *and* ***L.R.S.*** *designed and supervised the study.* ***R.S.J.*** *and* ***M.F.P.*** *performed biological sampling.* ***M.R.S.*** *performed statistical analysis.* Acknowledgements We thank the participating slaughterhouses and their inspection staff for their cooperation during sample collection. Data Availability The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. References Abuseir S, Epe C, Schnieder T, Klein G, Kühne M. 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Bovine cysticercosis epidemiology and the economic impact of the triceps brachii incision in a South African export abattoir. Prev Vet Med. 2023;220:106050. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9283419","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633440406,"identity":"9187aa48-1ff2-47af-b981-4087b0ca4686","order_by":0,"name":"Emilly Liandra Camargo Silva","email":"","orcid":"","institution":"Universidade Federal de Mato Grosso do Sul (UFMS)","correspondingAuthor":false,"prefix":"","firstName":"Emilly","middleName":"Liandra Camargo","lastName":"Silva","suffix":""},{"id":633440407,"identity":"82a159b4-29f2-471a-9b02-d627db074721","order_by":1,"name":"Lara Pael Coelho","email":"","orcid":"","institution":"Universidade Federal de Mato Grosso do Sul (UFMS)","correspondingAuthor":false,"prefix":"","firstName":"Lara","middleName":"Pael","lastName":"Coelho","suffix":""},{"id":633440408,"identity":"44342aa0-713a-45f8-a613-fa54cdc88365","order_by":2,"name":"Cristine K. Jéssica","email":"","orcid":"","institution":"Flábio Ribeiro de Araújo. Embrapa Beef Cattle","correspondingAuthor":false,"prefix":"","firstName":"Cristine","middleName":"K.","lastName":"Jéssica","suffix":""},{"id":633440409,"identity":"c9ce6a68-86c1-42b8-a5c7-f54080dc17a4","order_by":3,"name":"Ana Paula Figueiredo","email":"","orcid":"","institution":"Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Embrapa Beef Cattle","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Paula","lastName":"Figueiredo","suffix":""},{"id":633440410,"identity":"2152f908-e243-474f-9613-cf5847352167","order_by":4,"name":"Ana Clara Leonardo Araújo","email":"","orcid":"","institution":"Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Embrapa Beef Cattle","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Clara Leonardo","lastName":"Araújo","suffix":""},{"id":633440411,"identity":"f55ed3af-f2cd-4230-8566-e4d3fe0a7ed3","order_by":5,"name":"Raquel Soares Juliano","email":"","orcid":"","institution":"Embrapa Pantanal","correspondingAuthor":false,"prefix":"","firstName":"Raquel","middleName":"Soares","lastName":"Juliano","suffix":""},{"id":633440412,"identity":"86bcdf9a-c9db-4055-959b-99629c69e962","order_by":6,"name":"Fernando Almeida Borges","email":"","orcid":"","institution":"Universidade Federal de Mato Grosso do Sul (UFMS)","correspondingAuthor":false,"prefix":"","firstName":"Fernando","middleName":"Almeida","lastName":"Borges","suffix":""},{"id":633440413,"identity":"ca26ea7c-69ab-49fa-8d92-6c407597091c","order_by":7,"name":"Marcelo Fontes Pereira","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Marcelo","middleName":"Fontes","lastName":"Pereira","suffix":""},{"id":633440414,"identity":"f51adec6-2167-42d9-ab23-93aaa7a64ae9","order_by":8,"name":"Marcio Roberto Silva","email":"","orcid":"","institution":"Embrapa Dairy Cattle, Juiz de Fora","correspondingAuthor":false,"prefix":"","firstName":"Marcio","middleName":"Roberto","lastName":"Silva","suffix":""},{"id":633440415,"identity":"965f3821-649a-4f52-9969-6be402ccb231","order_by":9,"name":"Paulo Henrique Duarte Cançado","email":"","orcid":"","institution":"Embrapa Beef Cattle","correspondingAuthor":false,"prefix":"","firstName":"Paulo","middleName":"Henrique Duarte","lastName":"Cançado","suffix":""},{"id":633440416,"identity":"3b41496c-18aa-4376-8f64-2c55c812406f","order_by":10,"name":"Lenita Ramires Santos","email":"","orcid":"","institution":"Embrapa Beef Cattle","correspondingAuthor":false,"prefix":"","firstName":"Lenita","middleName":"Ramires","lastName":"Santos","suffix":""},{"id":633440417,"identity":"f6d26271-1548-43f4-a11a-812aaa911c63","order_by":11,"name":"Flábio Ribeiro Araújo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYBAC9nYGNoYEKMeAoQJEgMEBnFp4DqNoOUOsFjhgbCNGCzPzswcPKhjkzdvPGBR8nGcTbS59+NmDHwx38nFrYTM3SDjDYDjnTI6B4cxtabk7+9LMDXsYnlk24NBiz8xgJpHYxsA4gyHHwJh32+HcDWeAIjwMhw1w28L+DaTFfgb/G6CWOf+BWti/Sf7Bq4UHbEviDAmQLQ0HgFp4zKTx28JTDvSLRPIMiWcFhjOOJefu7OEpN5YxeIZbC3v7toc/KmxsZ/AnbzP4UGOXu52HfdvDNxV3cGqBAgkQwQZTxQaPHUKA+QFCyygYBaNgFIwCBAAARR9R5DubhK8AAAAASUVORK5CYII=","orcid":"","institution":"Embrapa Beef Cattle","correspondingAuthor":true,"prefix":"","firstName":"Flábio","middleName":"Ribeiro","lastName":"Araújo","suffix":""}],"badges":[],"createdAt":"2026-03-31 18:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9283419/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9283419/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108400148,"identity":"6ff4114f-0673-4f4b-a55e-9726430ed787","added_by":"auto","created_at":"2026-05-04 08:41:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":427854,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative analytical sensitivity of primer sets for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. saginata\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e detection.\u003c/strong\u003e (A) COX1 (428 bp): 0.48 ng; (B) LSU rRNA (328 bp): 0.97 ng; (C) COX1 (253 bp): 1.95 ng. White arrows indicate the last amplified dilution. 1 kb: DNA molecular weight marker.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-9283419/v1/a43c49e22c37e9618bc19bc9.png"},{"id":108803690,"identity":"38bc9e91-87de-4b2e-8c90-20a8e1077f8e","added_by":"auto","created_at":"2026-05-08 15:03:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":738926,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9283419/v1/898e99ad-9a8a-4603-8f29-7042421974d4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Beyond the Primer: Anatomical Site and Cyst Viability Drive the Molecular Detection of Taenia saginata in Slaughterhouse Samples","fulltext":[{"header":"Background","content":"\u003cp\u003eBrazil plays a prominent role in the global beef market, consolidating its position as the world\u0026rsquo;s largest beef exporter (ABIEC, 2025). In this context, strict hygienic\u0026ndash;sanitary control throughout the production chain is essential to ensure food safety and maintain access to international markets. Among parasitic diseases affecting both public health and the meat industry, bovine cysticercosis\u0026mdash;caused by the larval stage of the zoonotic cestode \u003cem\u003eTaenia saginata\u003c/em\u003e\u0026mdash;stands out as a major cause of partial or total carcass condemnation, leading to significant economic losses (Jorga et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The taeniasis\u0026ndash;cysticercosis complex represents a critical public health concern, directly involving the human\u0026ndash;animal interface and environmental contamination through the shedding of infective eggs (Eichenberger et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Elbarbary et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn Brazil, according to the Regulation of Industrial and Sanitary Inspection of Products of Animal Origin (RIISPOA) (Brasil, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), the diagnosis of bovine cysticercosis relies on post-mortem macroscopic identification, classifying cysticerci as viable or calcified. Although routine meat inspection is essential, visual examination presents inherent limitations. Degenerated or calcified cysticerci are notoriously difficult to distinguish from other pathological lesions. Consequently, misclassification frequently occurs, resulting in the overestimation of infection rates and the inappropriate condemnation of carcasses (El-Sayad et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent studies reinforce these diagnostic challenges. In the state of Tocantins, Brazil, visual inspection identified a low frequency of cystic lesions, yet molecular analysis and histopathology confirmed \u003cem\u003eT. saginata\u003c/em\u003e in only a fraction of these samples, identifying many as non-specific granulomatous lesions or hydatid disease (Figueiredo et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similarly, El-Sayad et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) demonstrated that visual inspection significantly overestimated the occurrence of \u003cem\u003eT. saginata\u003c/em\u003e cysticerci in cattle slaughtered in Egypt when compared to PCR confirmation. Together, these findings highlight the limited specificity of macroscopic diagnosis and emphasize the need for complementary approaches.\u003c/p\u003e \u003cp\u003eMolecular techniques, particularly polymerase chain reaction (PCR), are widely utilized for their high analytical sensitivity and specificity (Gonz\u0026aacute;lez et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Cuttell et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Uys et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Nevertheless, PCR performance is highly susceptible to field variables, including lesion stage, DNA degradation in mineralized cysts, and primer efficiency (Abuseir et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Uys et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe hypothesized that incorporating conventional PCR as a complementary tool would significantly improve the specificity of visual diagnosis, particularly in lesions classified as degenerated or calcified. Furthermore, we hypothesized that differences in primer sequences and the anatomical origin of the lesion could influence diagnostic reliability. Accordingly, this study aimed to evaluate the performance of previously described primer sequences against a novel primer set standardized herein. Assays were conducted using slaughterhouse samples from Mato Grosso do Sul, Brazil, to assess the practical applicability of PCR as a complementary diagnostic tool in routine meat inspection.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and sample collection\u003c/h2\u003e \u003cp\u003eThis study was conducted between April 2023 and June 2024 in the state of Mato Grosso do Sul, Brazil, to evaluate and compare the diagnostic performance of three conventional PCR assays for detecting \u003cem\u003eTaenia saginata\u003c/em\u003e DNA in bovine tissue. A total of 232 tissue samples were analyzed. The majority of the samples (n\u0026thinsp;=\u0026thinsp;224) were collected from cattle slaughtered at an establishment operating under the Federal Inspection Service (SIF), while the remaining samples (n\u0026thinsp;=\u0026thinsp;8) were sourced from a slaughterhouse under the State Inspection Service (SIE). All samples were collected at the Final Inspection Department from carcasses that had been diverted due to suspected cystic lesions identified during routine post-mortem examination. Tissue fragments were excised from standard anatomical inspection sites, including the heart, liver, diaphragm, masseter and pterygoid muscles, and esophagus. Each sample was individually cataloged (recording carcass number, batch number, anatomical location, and collection date), placed in a sterile plastic container, maintained under refrigeration, and transported to the Animal Health Laboratory at Embrapa Beef Cattle for molecular analysis. To ensure sufficient material for DNA extraction, cystic structures were collected along with a small margin of adjacent tissue.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMacroscopic classification\u003c/h3\u003e\n\u003cp\u003eMacroscopic classification and subsequent carcass disposition strictly adhered to the criteria established by Brazilian sanitary inspection regulations, which define infection severity based on the number and anatomical distribution of the cysts. Based on slaughterhouse records and visual inspection, the 232 collected samples were divided into three distinct categories: calcified cysts (n\u0026thinsp;=\u0026thinsp;180, which included all eight samples from the state-inspected slaughterhouse), viable cysts (n\u0026thinsp;=\u0026thinsp;12), and visually negative or inconclusive lesions (n\u0026thinsp;=\u0026thinsp;40). This last group served as specificity controls and comprised non-specific macroscopic findings, such as hepatic fatty nodules, necrotic or inflammatory liver nodules, uncharacterized calcified liver nodules, unspecified cardiac nodules, focal connective tissue in the liver and heart, and hepatic sarcocystosis lesions.\u003c/p\u003e\n\u003ch3\u003eDNA extraction and quality assessment\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from approximately 25 mg of homogenized tissue using the PureLink Genomic DNA Mini Kit (Invitrogen, USA), according to the manufacturer\u0026rsquo;s instructions. DNA concentration and purity were determined using a NanoDrop 2000c spectrophotometer (Thermo Scientific, USA). DNA integrity was verified by electrophoresis on a 0.8% agarose gel.\u003c/p\u003e\n\u003ch3\u003ePCR assays\u003c/h3\u003e\n\u003cp\u003eThree conventional PCR assays for the detection of \u003cem\u003eT. saginata\u003c/em\u003e were evaluated. The first assay, designed and standardized in this study, targeted a 428-base pair (bp) fragment of the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene. Specific primers were designed using the Primer-BLAST tool (NCBI): forward (5\u0026prime;-GGATTGCCTCGTCGTGTTTG-3\u0026prime;) and reverse (5\u0026prime;-CTAAGCATGATGCAAAAGGC-3\u0026prime;). Amplification reactions (20 \u0026micro;L) contained GoTaq Colorless Master Mix 2X (Promega, USA), 0.25 \u0026micro;M of each primer, and 200 ng of template DNA. Thermal cycling was performed in an MJ Mini Personal Thermal Cycler (Bio-Rad, USA), consisting of an initial denaturation at 95\u0026deg;C for 10 min, followed by 35 cycles of 94\u0026deg;C for 1 min, 51\u0026deg;C for 1 min, and 72\u0026deg;C for 40 s, with a final extension step at 72\u0026deg;C for 7 min.\u003c/p\u003e \u003cp\u003eThe second assay, previously described by Jardim et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), targeted a 328-bp fragment of the large subunit (LSU) rRNA gene using the primers TBR-3 (generic forward, 5\u0026rsquo;-GGCTTGTTTGAATGGTTTGACG-3\u0026rsquo;) and TBR-4 (\u003cem\u003eT. saginata\u003c/em\u003e-specific reverse, 5\u0026rsquo;-CGACTCATGAAGATAAACAAGGT-3\u0026rsquo;). The PCR reactions (20 \u0026micro;L) were performed using the same reagent concentrations as the first assay. The cycling program consisted of an initial denaturation at 94\u0026deg;C for 2 min, followed by 40 cycles of 94\u0026deg;C for 30 s, 59\u0026deg;C for 30 s, and 72\u0026deg;C for 1 min, concluding with a final extension at 72\u0026deg;C for 5 min.\u003c/p\u003e \u003cp\u003eThe third assay, adapted from El-Dakhly et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), amplified a 253-bp fragment of the COX1 gene using the forward (5\u0026prime;-GGGTGCTGGTATAGGGTGGACT-3\u0026prime;) and reverse (5\u0026prime;-ACGTAAATAAATAAGCCCACAATATT-3\u0026prime;) primers. Amplification reactions (25 \u0026micro;L) contained GoTaq Colorless Master Mix 2X, 0.3 \u0026micro;M of each primer, 200 ng of template DNA, and nuclease-free water to reach the final volume. The cycling conditions included an initial denaturation at 94\u0026deg;C for 4 min, followed by 35 cycles of 94\u0026deg;C for 30 s, 59\u0026deg;C for 1 min, and 72\u0026deg;C for 1 min, with a final extension step at 72\u0026deg;C for 7 min.\u003c/p\u003e \u003cp\u003eTo assess the presence of PCR inhibitors in samples that tested negative for \u003cem\u003eT. saginata\u003c/em\u003e, an internal control PCR targeting the bovine \u003cem\u003eDGAT1\u003c/em\u003e gene was performed. This primer pair amplified a 282 bp fragment of this constitutive gene, confirming DNA quality and amplification competence. PCR products from all assays were separated by electrophoresis on 1.5% agarose gels in 1\u0026times; TBE buffer at 100\u0026ndash;120 V. A 1 kb Plus DNA ladder (Invitrogen, USA) was used as a molecular size marker. Gels were stained with a commercial nucleic acid dye and visualized under ultraviolet light.\u003c/p\u003e\n\u003ch3\u003eAnalytical sensitivity assessment\u003c/h3\u003e\n\u003cp\u003eThe analytical sensitivity of the standardized assay was evaluated using a 1:2 serial dilution of DNA extracted from viable \u003cem\u003eT. saginata\u003c/em\u003e cysticerci (initial concentration of 200 ng/\u0026micro;L). The detection limit was defined as the lowest concentration that produced a clear, reproducible band on the 1.5% agarose gel.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eTo evaluate the detection capability across different molecular targets, the positivity frequencies of three PCR primer sets were analyzed: COX1 (428 bp; designed in this study), LSU rRNA (328 bp), and a second COX1 target (253 bp). To determine if PCR positivity was significantly associated with the macroscopic classification of the lesions (viable cysts, calcified cysts, or other lesions), the Chi-square test or Fisher's Exact test was employed, depending on the expected cell frequencies. Furthermore, because the same 232 samples were evaluated across all three molecular targets (paired data), Cochran's Q test was used to assess whether there was a statistically significant difference in overall diagnostic sensitivity among the three primers. All statistical analyses were performed using R software version 4.3.2 (R Core Team, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A significance level of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was adopted for all analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eEvaluation of analytical sensitivity demonstrated the following detection limits: 0.48 ng for the COX1 (428 bp) primer pair, 0.97 ng for LSU rRNA (328 bp), and 1.95 ng for COX1 (253 bp). These results indicate that the novel COX1 (428 bp) assay yielded the highest analytical sensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMolecular detection of \u003cem\u003eT. saginata\u003c/em\u003e DNA varied significantly depending on the macroscopic developmental stage of the lesion, confirming the profound impact of cyst degeneration on PCR performance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). For viable cysts (n\u0026thinsp;=\u0026thinsp;12), positivity rates were high, reaching 50.0% for COX1 (428 bp), 58.3% for LSU rRNA (328 bp), and 66.7% for COX1 (253 bp). In contrast, detection in calcified cysts (n\u0026thinsp;=\u0026thinsp;180) dropped substantially to 26.7%, 30.0%, and 28.9%, respectively, reflecting severe DNA degradation in chronic lesions. Samples macroscopically classified as 'other lesions' (n\u0026thinsp;=\u0026thinsp;40) exhibited baseline positivity rates of only 2.5% to 5.0%, highlighting the high specificity of the assays.\u003c/p\u003e \u003cp\u003eWhen comparing the overall diagnostic performance of the three molecular targets across the 232 paired samples, the LSU rRNA target yielded the highest absolute number of positive detections (27.1%), followed by the 253 bp COX1 target (26.3%) and the 428 bp COX1 target (23.7%). However, Cochran's Q test revealed that these variations were not statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating that no single molecular target demonstrated absolute diagnostic superiority over the others in routine field samples (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of PCR positivity across three molecular targets for the detection of \u003cem\u003eTaenia saginata\u003c/em\u003e according to macroscopic lesion stage.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample classification\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCOX1 (428 bp) [This study] Pos\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCOX1 (428 bp) [This study] Neg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLSU rRNA (328 bp) [Jardim et al.] Pos\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLSU rRNA (328 bp) [Jardim et al.] Neg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCOX1 (253 bp) [El-Dakhly et al.] Pos\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCOX1 (253 bp) [El-Dakhly et al.] Neg\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCalcified cyst\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eViable cyst\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther lesions*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e232\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e55\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e177\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e63\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e169\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e61\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e171\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003e*Includes macroscopic findings such as various hepatic nodules (fatty, necrotic, inflammatory, calcified, and unspecified), cardiac nodules, connective tissue in the liver and heart, and hepatic sarcocystosis.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eCrucially, organ-stratified analysis revealed that anatomical location profoundly impacts molecular confirmation rates, particularly for chronic lesions (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Among calcified cysts, samples from the liver (n\u0026thinsp;=\u0026thinsp;105) exhibited drastically reduced PCR positivity (11.4% to 15.2%). In sharp contrast, calcified cysts in the heart (n\u0026thinsp;=\u0026thinsp;50) and head muscles (n\u0026thinsp;=\u0026thinsp;20) demonstrated substantially higher molecular positivity rates (46.0%\u0026ndash;54.0% and 50.0%\u0026ndash;60.0%, respectively). For viable cysts, found exclusively in the heart and masseter muscle, detection rates remained consistently high (66.7% to 77.8%). This marked discrepancy suggests that visual inspection of the liver suffers from low specificity (due to misclassification of non-parasitic granulomas) or that the hepatic microenvironment accelerates the degradation of parasite DNA (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of PCR positivity for \u003cem\u003eTaenia saginata\u003c/em\u003e across three molecular targets according to organ and macroscopic lesion stage.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrgan\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCyst Type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCOX1 (428 bp) Pos\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCOX1 (428 bp) Neg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLSU rRNA (328 bp) Pos\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLSU rRNA (328 bp) Neg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCOX1 (253 bp) Pos\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCOX1 (253 bp) Neg\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCalcified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeart\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCalcified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeart\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eViable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMasseter Muscle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCalcified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMasseter Muscle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eViable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePterygoid Muscle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCalcified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiaphragm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCalcified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMixed Samples*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCalcified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTOTAL\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e192\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e60\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e132\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e54\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e138\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e61\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e131\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003e*Mixed samples represent combined adjacent tissues occasionally collected from highly calcified or diffuse areas.\u003c/em\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRoutine post-mortem inspection of bovine carcasses remains the primary line of defense against the transmission of \u003cem\u003eT. saginata\u003c/em\u003e (Scandrett et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, the subjective nature of macroscopic evaluation often leads to diagnostic uncertainty, particularly with degenerated or calcified lesions (El-Sayad et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The present study evaluated the diagnostic performance of three conventional PCR assays, revealing that while molecular tools are highly specific, their sensitivity in the field is profoundly dictated by the developmental stage of the cyst and its anatomical location.\u003c/p\u003e \u003cp\u003eConsistent with previous literature (Costa et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), our results demonstrated a significant decline in PCR positivity as cysticerci degenerate. Viable cysts contain intact parasitic cells with highly preserved DNA, making them ideal targets. In contrast, calcification represents the end-stage of a severe host-parasite inflammatory response (Peixoto et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Macroscopically and histologically, degenerated cysts are characterized by caseous necrosis, severe cellular infiltration, and eventual mineralization, culminating in the loss of identifiable parasitic features (Elbarbary et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This caseous necrosis physically entraps remaining structures and severely degrades nucleic acids. Consequently, the lower molecular detection rate in calcified lesions represents a biological limitation of the sample rather than a failure of the PCR assays. The high specificity of the assays was corroborated by the minimal positivity (2.5%\u0026ndash;5.0%) observed in the \"other lesions\" category (Gonz\u0026aacute;lez et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, no statistically significant difference in diagnostic performance was observed among the three molecular targets evaluated (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This finding is highly relevant, indicating that the current bottleneck in the molecular confirmation of bovine cysticercosis is the intrinsic biological degradation of the target DNA, not primer design. It also validates the newly designed COX1 (428 bp) primers as a reliable, interchangeable tool for future epidemiological studies.\u003c/p\u003e \u003cp\u003eThe most striking and novel finding of this study emerged from the organ-stratified analysis. The profound discrepancy in molecular confirmation between calcified cysts in the liver (11.4%\u0026ndash;15.2%) and those in the heart and head muscles (46.0%\u0026ndash;60.0%) provides a critical biological explanation for the low agreement between visual inspection and PCR. This stark contrast supports two primary hypotheses. First, visual inspection of the liver suffers from inherently low specificity. As a major filtering organ, the liver is susceptible to various pathological insults resulting in granulomatous and calcified lesions (e.g., hepatic fascioliasis, hydatidosis, bacterial abscesses). During the rapid pace of an industrial slaughter line, these non-parasitic granulomas are easily misclassified as calcified \u003cem\u003eC. bovis\u003c/em\u003e (Costa et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese observations align with previous reports analyzing the macroscopic profile of cysticercosis. Peixoto et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) highlighted that hepatic cysticerci are predominantly non-viable (71.0% to 87.5%). Furthermore, Costa et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) emphasized that non-viable cysticercus lesions are easily confused with other etiologies, reducing the specificity of gross anatomical examination. This explains why high visual occurrence rates of hepatic cysticerci often correspond to low true-positive rates upon histological or molecular confirmation (Peixoto et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSecond, distinct tissue microenvironments likely dictate DNA preservation. The liver is an enzymatically aggressive environment with a robust local immune system (e.g., Kupffer cells). It is highly probable that the clearance of parasitic DNA occurs at a much faster rate in the hepatic parenchyma than in striated muscle tissue, where calcified granulomas may retain remnant DNA for extended periods.\u003c/p\u003e \u003cp\u003eThese findings have direct economic implications for the global beef industry. Relying solely on macroscopic inspection for hepatic lesions leads to a massive overestimation of bovine cysticercosis, resulting in the unnecessary condemnation or costly cold-treatment of carcasses possessing only non-specific hepatic scars. By demonstrating that the vast majority of PCR-negative \"calcified cysts\" are clustered in the liver, this study advocates for a rigorous reevaluation of sanitary condemnation criteria.\u003c/p\u003e \u003cp\u003eDespite these robust findings, this study has certain limitations that warrant consideration. The relatively low number of viable cysts (n\u0026thinsp;=\u0026thinsp;12) compared to calcified lesions (n\u0026thinsp;=\u0026thinsp;180) reflects the natural frequency of chronic infections encountered in slaughterhouse lines, but restricts broader statistical inferences regarding early-stage cysticerci. Additionally, the initial sample screening relied on the routine visual inspection conducted by local abattoir staff. While this accurately reflects real-world industry practices and validates the need for molecular confirmation, it inherently carries the subjectivity of visual triaging. Future studies incorporating randomized carcass sampling regardless of gross macroscopic findings could further elucidate the true baseline prevalence of \u003cem\u003eT. saginata\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eConventional PCR is a highly specific complementary tool for diagnosing bovine cysticercosis, though its sensitivity is intrinsically limited by the severe DNA degradation characteristic of calcified cysts. Our findings validate the newly designed COX1 (428 bp) primer set as a reliable alternative to previously established targets.\u003c/p\u003e \u003cp\u003eCrucially, this study reveals that the anatomical location of a lesion dictates molecular detection rates. The vast majority of diagnostic discrepancies between visual inspection and PCR originate in the liver, strongly suggesting that macroscopic examination of hepatic tissue significantly overestimates the prevalence of \u003cem\u003eT. saginata\u003c/em\u003e due to the misclassification of non-parasitic granulomas. To improve epidemiological surveillance and mitigate the economic impact of unwarranted carcass condemnations, we strongly recommend that future molecular confirmation efforts\u0026mdash;and routine sanitary evaluations\u0026mdash;prioritize suspect lesions identified in the heart and striated muscles over those found in the liver.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNo animals were slaughtered specifically for the purposes of this study. All biological samples were obtained from cattle processed for commercial purposes under official sanitary inspection. Sample collection occurred during routine post-mortem inspection procedures at federally and state-inspected abattoirs, in accordance with Brazilian legislation governing the industrial and sanitary inspection of products of animal origin (Decree No. 9,013/2017 and subsequent amendments). Because the study was based exclusively on materials collected during routine inspection and did not involve experimental intervention or live animal handling, approval by an institutional animal ethics committee was not required.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was financed in part by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES), Brazil \u0026ndash; Finance Code 001. This research was also funded by the project \u0026ldquo;Beef Cattle National Science and Technology Institute,\u0026rdquo; supported by the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) (Grant No. 408696/2024-9), and by CNPq Grants No. 440559/2022-7 and 315857/2021-8.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e***E.L.C.S., L.P.C., J.C.K.M., A.P.F.,*** *and* ***A.C.L.A.*** *conducted the experiments.* ***F.R.A., F.A.B., P.H.D.C.,*** *and* ***L.R.S.*** *designed and supervised the study.* ***R.S.J.*** *and* ***M.F.P.*** *performed biological sampling.* ***M.R.S.*** *performed statistical analysis.*\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank the participating slaughterhouses and their inspection staff for their cooperation during sample collection.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbuseir S, Epe C, Schnieder T, Klein G, K\u0026uuml;hne M. Visual diagnosis of \u003cem\u003eTaenia saginata\u003c/em\u003e cysticercosis during meat inspection: is it unequivocal? Parasitol Res. 2006;99(4):405\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e(2025). \u003cem\u003eBeef Report 2025: Perfil da Pecu\u0026aacute;ria no Brasil\u003c/em\u003e. ABIEC, Associa\u0026ccedil;\u0026atilde;o Brasileira das Ind\u0026uacute;strias Exportadoras de Carnes (ABIEC), S\u0026atilde;o Paulo B. Available online: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://abiec.com.br/publicacoes/beef-report-2025-perfil-da-pecuaria-no-brasil/\u003c/span\u003e\u003cspan address=\"https://abiec.com.br/publicacoes/beef-report-2025-perfil-da-pecuaria-no-brasil/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrasil. que disp\u0026otilde;em sobre a inspe\u0026ccedil;\u0026atilde;o industrial e sanit\u0026aacute;ria de produtos de origem animal. Brazil: Di\u0026aacute;rio Oficial da Uni\u0026atilde;o, Bras\u0026iacute;lia; 2017. Decreto n\u0026ordm; 9.013, de 29 de mar\u0026ccedil;o de 2017. Regulamenta a Lei n\u0026ordm; 1.283, de 18 de dezembro de 1950, e a Lei n\u0026ordm; 7.889, de 23 de novembro de 1989.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosta AD, Pires PS, Pinto PSA, Guimar\u0026atilde;es-Peixoto RPM, Bevilacqua PD. Morphological and evolutionary characteristics of cysticercus from Taenia saginata and their influence on the macroscopic diagnosis. Revista Brasileira de Parasitol Veterin\u0026aacute;ria. 2012;21(2):150\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuttell L, Owen H, Lew-Tabor AE, Traub RJ. Bovine cysticercosis\u0026ndash;development of a real-time PCR to enhance classification of suspect cysts identified at meat inspection. Vet Parasitol. 2013;194(1):65\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEichenberger RM, Thomas LF, Gabri\u0026euml;l S, Bobić B, Devleesschauwer B, Robertson LJ, Saratsis A, Torgerson PR, Braae UC, Dermauw V, et al. Epidemiology of Taenia saginata taeniosis/cysticercosis: a systematic review of the distribution in East, Southeast and South Asia. Volume 13. Parasites \u0026amp; Vectors; 2020. p. 234.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElbarbary NK, Gareh A, Abdelhaseib M, Fotouh A, Abdelmotilib NM, Ragab MF, Dandrawy MK. \u003cem\u003eCysticercus bovis\u003c/em\u003e in slaughtered cattle in upper Egypt: implications for food safety. BMC Vet Res. 2025;21:344.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Dakhly KM, Hany SA, Arafa WM, Abdel-Fatah OR, Abdel-Atty NS, El-Nahass ES. The prevalence and molecular detection of bovine cysticercosis and its impact on slaughtered cattle in Egypt. J Parasitic Dis. 2023;47:527\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Sayad MH, Farag H, El-Taweel H, Fadly R, Salama N, Ahmed AAE, El-Latif NFA. \u003cem\u003eCysticercus bovis\u003c/em\u003e in cattle slaughtered in North Egypt: Overestimation by the visual inspection method. Veterinary World. 2021;14:155\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFigueiredo BNS, Lib\u0026oacute;rio RA, Sato M, da Silva CF, Pereira-Junior RA, Chigusa Y, Kawai S, Sato MO. Occurrence of Bovine Cysticercosis in Two Regions of the State of Tocantins-Brazil and the Importance of Pathogen Identification. Pathogens. 2019;8:66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonz\u0026aacute;lez LM, Villalobos N, Montero E, Morales J, Sanz RA, Muro A, Harrison LJ, Parkhouse RM, G\u0026aacute;rate T. Differential molecular identification of Taeniid spp. and Sarcocystis spp. cysts isolated from infected pigs and cattle. Vet Parasitol. 2006;142(1\u0026ndash;2):95\u0026ndash;101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJardim EAG, Linhares GFC, Torres FAG, Ara\u0026uacute;jo JDB, Barbosa SM. Diferencia\u0026ccedil;\u0026atilde;o espec\u0026iacute;fica entre Taenia saginata e Taenia solium por ensaio de PCR e duplex-PCR. Ci\u0026ecirc;ncia Rural. 2006;36:166\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJorga E, Van Damme I, Mideksa B, Zewde D, Gabri\u0026euml;l S. Improved estimation of the prevalence of bovine cysticercosis and the diagnostic test characteristics in the absence of a reference standard using Bayesian Latent Class models, the example of Jimma and Ambo abattoirs, Ethiopia. Volume 66. Veterinary Parasitology: Regional Studies and Reports; 2025. p. 101365.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeixoto RPMG, Pinto PSA, Santos TO, Silva LF, Acevedo-Nieto EC, Silva AR. Perfil da implanta\u0026ccedil;\u0026atilde;o de cisticercos de Taenia saginata em s\u0026iacute;tios musculares n\u0026atilde;o usuais e sua import\u0026acirc;ncia para a Sa\u0026uacute;de P\u0026uacute;blica. Pesquisa Veterin\u0026aacute;ria Brasileira. 2018;38(1):23\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR Core Team. (2023). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Available online: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.R-project.org/\u003c/span\u003e\u003cspan address=\"https://www.R-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScandrett B, Parker S, Forbes L, Gajadhar A, Dekumyoy P. Distribution of \u003cem\u003eTaenia saginata\u003c/em\u003e cysticerci in tissues of experimentally infected cattle. Vet Parasitol. 2012;189(2\u0026ndash;4):261\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUys M, Fosgate GT, Seguino A. Bovine cysticercosis epidemiology and the economic impact of the triceps brachii incision in a South African export abattoir. Prev Vet Med. 2023;220:106050.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bovine cysticercosis, Taenia saginata, PCR, Molecular diagnosis, Meat inspection, Food safety","lastPublishedDoi":"10.21203/rs.3.rs-9283419/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9283419/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBovine cysticercosis, caused by the larval stage of \u003cem\u003eTaenia saginata\u003c/em\u003e, results in significant economic losses for the beef industry due to carcass condemnation. While routine post-mortem inspection is mandatory, visual diagnosis lacks specificity, particularly for degenerated or calcified cysts. This study evaluated the diagnostic performance of three conventional PCR assays\u0026mdash;a widely used LSU rRNA target (328 bp), a previously described COX1 target (253 bp), and a novel COX1 target (428 bp) designed herein\u0026mdash;as complementary tools to visual inspection. A total of 232 suspect macroscopic lesions (180 calcified cysts, 12 viable cysts, and 40 non-specific lesions) were collected from cattle slaughtered in Mato Grosso do Sul, Brazil.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur results demonstrated no statistically significant difference in diagnostic sensitivity among the three primer sets (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating that molecular confirmation is not limited by primer selection. Instead, molecular detection was profoundly driven by cyst viability and anatomical location (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). While viable cysts yielded high positivity rates (up to 66.7%), detection in calcified lesions dropped to approximately 28.9% due to severe DNA degradation. Crucially, calcified hepatic lesions exhibited drastically lower PCR positivity (11.4%\u0026ndash;15.2%) compared to those in the heart (46.0%\u0026ndash;54.0%) and head muscles (50.0%\u0026ndash;60.0%).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings indicate that visual inspection of hepatic tissue significantly overestimates the prevalence of \u003cem\u003eT. saginata\u003c/em\u003e, likely due to the misclassification of non-parasitic granulomas. To improve diagnostic accuracy and prevent unwarranted carcass condemnations, molecular confirmation efforts and routine sanitary evaluations should prioritize suspect lesions in the heart and striated muscles over those found in the liver.\u003c/p\u003e","manuscriptTitle":"Beyond the Primer: Anatomical Site and Cyst Viability Drive the Molecular Detection of Taenia saginata in Slaughterhouse Samples","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 08:41:13","doi":"10.21203/rs.3.rs-9283419/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-12T07:13:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T04:26:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T19:05:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T15:45:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50727887993772283331798832516576985786","date":"2026-04-30T03:11:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9488388447786514947159243727729112261","date":"2026-04-29T19:48:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T14:47:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1855477290958217510954248812541810723","date":"2026-04-27T18:50:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"285826930823696373875194381442557514460","date":"2026-04-27T14:18:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77320107453580127554121733913148025012","date":"2026-04-22T18:37:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-22T18:27:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-22T18:24:30+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-20T16:52:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-18T16:04:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2026-04-18T15:59:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8d291bac-94d7-4911-91b9-ffd32fcff170","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-12T07:13:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T04:26:05+00:00","index":68,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T19:05:19+00:00","index":67,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T15:45:23+00:00","index":66,"fulltext":""},{"type":"reviewerAgreed","content":"50727887993772283331798832516576985786","date":"2026-04-30T03:11:22+00:00","index":65,"fulltext":""},{"type":"reviewerAgreed","content":"9488388447786514947159243727729112261","date":"2026-04-29T19:48:53+00:00","index":64,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T14:47:05+00:00","index":63,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T07:29:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 08:41:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9283419","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9283419","identity":"rs-9283419","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}