Optimizing diagnostic sites for FMDV carrier identification: A systematic evaluation of tissue tropism in goats

Introduction

Foot-and-mouth disease virus is recognized for its ability to establish a persistent infection in ruminants, a condition defined by the presence of infectious virus in the oropharyngeal region for 28 days or more post-infection (Jena, 2022) [14] . While hematobiochemical alterations during the acute phase of FMDV infection have been documented in goats, showing significant decreases in hemoglobin, total erythrocytes, packed cell volume, and total protein, there remains a paucity of literature regarding the anatomical sites of viral persistence in the carrier state. The mechanisms of viral persistence have been extensively documented in cattle, research regarding the anatomic localization and pathogenesis of the carrier state in goats remains limited (Jena, 2022; Sarry et al., 2023) [14,22] . Addressing this knowledge gap is essential, as persistent infection occurs across small ruminants regardless of vaccination history or initial clinical presentation (Pacheco et al., 2015) [20] . Unlike cattle, where viral persistence is primarily localized to the nasopharyngeal mucosa, the specific tissue tropism in caprine species have not been systematically characterized (Conrady et al., 2024; Stenfeldt & Arzt, 2020) [7,8,24] . This study addresses this discrepancy by molecular and morphological findings to determine which tissues, serve as primary reservoirs for viral persistence in goats (Henning et al., 2025; Stenfeldt et al., 2019) [13,28] . Furthermore, this investigation aims to elucidate whether goat tissue tropism aligns more closely with the nasopharyngeal reservoirs observed in bovines or the tonsillar crypts identified in ovine models (Conrady et al., 2024) [7,8] .

Materials and Methods

Study Area and Experimental Animals

The research was conducted in a region endemic to Footand-mouth disease virus, allowing for the evaluation of goats under natural infection conditions. The study utilized a cohort of 60 goats. To investigate the subclinical carrier state, the animals were monitored to confirm they met the formal definition of an FMDV carrier: the persistence of the viral genome in the oropharyngeal region for 28 days or more following the initial infection). This phase is distinct from the active phase of infection, during which goats typically exhibit significant systemic alterations in hematobiochemical profiles, such as decreased hemoglobin and total protein.

Tissue Collection and Processing

Systemic post-mortem examinations were conducted to harvest 11 distinct anatomical tissues. To preserve the integrity of the viral RNA, samples from the nasopharynx, soft palate, and lymphoreticular tissues were collected using sterile techniques. Tissues were collected in sterile tubes containing transport media, such as phosphate-buffered glycerol with antibiotics, to stabilize the virus prior to laboratory analysis. Tissue specimens intended for morphological analysis were fixed in a 10% neutral buffered formalin solution ((Bancroft and Gamble, 2008) [3] . The following eleven tissue types were evaluated: Middle tongue, Ventral soft palate, Dorsal soft palate, Dorsal nasopharynx 1(Cranial), Dorsal nasopharynx 2(Caudal), Heart, Retropharyngeal lymph node, Palatine tonsil, Prescapular lymph node, Prefemoral lymph node, Mandibular lymph node

Molecular Protocols and Instrumentation

The primary diagnostic tool for identifying the viral reservoirs was real-time reverse transcriptase PCR (rRT-PCR), which provides the sensitivity required to detect lowlevel viral persistence in carrier tissues (Arzt et al., 2017; Pacheco et al., 2015) [24,20,25] . Total RNA was extracted from the 11 tissue types using standardized lysis and purification protocols. The rRT-PCR assays targeted the highly conserved 3D polymerase region of the FMDV genome (Stenfeldt et al., 2015) [25] .

Histopathological Analysis

To characterize the micro-anatomical localization of the virus and any associated cellular changes, fixed tissues were processed for histopathology. Tissues were dehydrated through graded alcohols, cleared, and embedded in paraffin blocks. Sections were cut at a thickness of 5 micrometer and stained with Hematoxylin and Eosin to allow for the assessment of epithelial changes and lymphoid activity within the sampled niches.

Software and Statistical Analysis

Data acquired from the real-time PCR system were analyzed to determine the percentage of positivity across the 11 tissue types, facilitating the identification of tissue tropism. To identify and prioritize the primary anatomical reservoirs of FMDV, a Pareto analysis was performed. This statistical technique was used to distinguish the 'vital few' tissues that contribute the most to overall viral detection from the 'trivial many' that offer low diagnostic yield.

Results and Discussion

The combined findings of the study were discussed with their significance in the context of established foot-andmouth disease virus pathogenesis models and diagnostic surveillance.

Tissue-Specific Distribution and Viral Load

Molecular analysis using rRT-PCR demonstrated that the highest rates of viral detection occurred in the nasopharyngeal region. The Dorsal Nasopharynx 1 exhibited the highest positivity rate at 25.00% (13/52), followed by the Dorsal Nasopharynx 2 at 23.08% (12/52). Significant viral persistence was also localized to the Dorsal Soft Palate (19.23%) and the Ventral Soft Palate (17.31%). In stark contrast, tissues that are traditionally prioritized in small ruminant surveillance, such as the Palatine Tonsils, yielded a positivity rate of only 1.92% (1/52). Peripheral sites, including the mandibular lymph nodes (1.92%), heart (3.85%), and tongue (7.69%), showed minimal involvement in long-term viral maintenance. The mean Ct values for tissue clustered between 24 and 38, which is characteristic of the low-level replication observed in carrier animals. It serves as a molecular hallmark of the Foot-and-mouth disease virus carrier state. The detection of Ct values in the 24-38 range provides evidence of low-level genomic maintenance rather than the robust replication seen in clinical cases. This research indicates that Ct values in FMDV carriers are "generally very high, "which is "indicative of low levels of FMDV RNA" within the pharyngeal tissues. This specific range reflects several critical biological and diagnostic realities of subclinical infection. This localization is particularly noteworthy because, while these subclinical carriers often exhibit normalized hematobiochemical profiles, recovering from the acute-phase reductions in hemoglobin and total protein, they continue to harbour the virus in these specific mucosal sites.

Anatomical Discrepancy

A primary contribution of this study is the resolution of the anatomical discrepancy between bovine and ovine persistence models as they relate to goats. Historically, goats have been grouped with sheep under the small ruminant diagnostic umbrella, which identifies the epithelial crypts of the oropharyngeal tonsils as the primary viral reservoir (Conrady et al., 2024; Stenfeldt et al., 2019) [7,8,28] . However, our results proved that caprine tissue tropism significantly diverged from the ovine tonsillar model. If veterinary practitioners were to rely solely on tonsillar sampling, they would have failed to identify over 98% of the carrier animals in this study (Conrady et al., 2024) [7,8] . Instead, the findings revealed that goats mirror the bovine model of persistence. In cattle, FMDV is primarily restricted to the nasopharyngeal mucosa, specifically the dorsal nasopharynx and the superior surface of the soft palate (Pacheco et al., 2015; Stenfeldt et al., 2015) [20,25] . By identifying the dorsal nasopharynx and soft palate as the definitive reservoirs in goats, this study suggests that the biological mechanisms of persistence in the caprine species are more closely aligned with those of large ruminants than with sheep (Arzt et al., 2017) [24] .

Pareto Analysis and Diagnostic Optimization

A Pareto analysis was applied to the tissue distribution data to prioritize diagnostic targets, revealing that four primary sites DNP1, DNP2, DSP, and VSP accounted for 84.62% of all positive detections (Table I). This distribution adheres to the "80/20 rule," where the "vital few" tissues provide the majority of the diagnostic yield. As shown in Graph 1, the steep initial slope of the cumulative line indicates that viral persistence is highly concentrated in these anatomical niches, while the flattening of the curve toward peripheral tissues (lymph nodes and heart) suggests they contribute negligible value to identifying the carrier state. This concentration of viral RNA confirms that goats mirror the bovine model of nasopharyngeal tropism (Arzt et al., 2017; Pacheco et al., 2015) [20,25] rather than the ovine model, which focuses on the oropharyngeal tonsils (Conrady et al., 2024; Stenfeldt et al., 2019) [7,8,28] . By prioritizing the "vital few" sites identified by the Pareto graph, surveillance protocols in endemic regions can be optimized for maximum sensitivity and efficiency (Conrady et al., 2024; Mardones et al., 2010) [7,8] . This targeted approach is essential for identifying subclinical carriers and preventing the silent circulation of endemic serotypes (Jena, 2022; Khan et al., 2024) [14] .

Histopathological alterations

Histopathological examination of the primary reservoirs, the dorsal nasopharynx and soft palate provided morphological evidence for this tissue tropism. Consistent subepithelial lymphoid activity was observed, characterized by welldefined lymphoid aggregations with follicular hyperplasia and a dense population of lymphocytes and macrophages (Fig. 1 to Fig. 8). In the DNP, the epithelium exhibited a mosaic appearance, where infected cells clustered together in small groups surrounded by healthy keratinocytes. This non-uniformity in the basal layer suggests the virus specifically targets large, polygonal cells, likely basal keratinocytes or dendritic-like cells (Pacheco et al., 2015; Stenfeldt et al., 2015) [20,25] . In the soft palate tissues, acantholytic-like changes were noted, marked by the loss of intercellular attachments within the stratified squamous layer. The infiltration of mononuclear cells from underlying follicles into the epithelium created a moth-eaten" appearance at the basal boundary. Conversely, the tongue showed hydropic or ballooning degeneration of prickle cells, while the myocardium maintained a regular arrangement of fibers with distinct striations, consistent with the subclinical nature of the persistent phase (Parthiban et al., 2015).

Conclusion

In conclusion, these results demonstrated that the dorsal nasopharynx and soft palate are the primary anatomical reservoirs for FMDV in goats. By proving that goats follow the bovine nasopharyngeal model rather than the ovine tonsillar model, this study provides a robust, data-driven foundation for refining species-specific diagnostic protocols and improving the efficacy of FMDV control programs in small ruminant populations.

Acknowledgement

AcknowledgmentsThe authors are grateful to the College of Veterinary Science & Animal Husbandry, Birsa Agricultural University, Kanke, Ranchi, for providing the necessary infrastructure and facilities to conduct this research. We also extend our sincere thanks to the ICAR-International Centre for Foot and Mouth Disease for their technical guidance, laboratory support, and expertise in viral diagnostics, which were essential to the completion of this study.

References

1. Arzt J, Pacheco JM, Stenfeldt C, Rodríguez LL. Pathogenesis of virulent and attenuated foot-and-mouth disease virus in cattle. Virol J. 2017 Mar 31;14:123.
2. Bamouh Z, Fakri F, Jazouli M, Safini N, Tadlaoui KO, Elharrak M. Peste des petits ruminants pathogenesis on experimental infected goats by the Moroccan 2015 isolate. BMC Vet Res. 2019 Nov 4;15:352.
3. Bancroft JD, Gamble M, editors. Theory and Practice of Histological Techniques. 6th ed. Edinburgh: Churchill Livingstone Elsevier; 2008.
4. Bandaw T, Fesseha H, Muluneh A, Mengistu TS, Kebede IA. Seroprevalence and molecular detection of foot and mouth disease virus in cattle in selected districts of Wolaita Zone, Southern Ethiopia. Sci Rep. 2024 Mar 25;14:7929.
5. Bertram MR, Vu LT, Pauszek SJ, Brito B, Hartwig EJ, Smoliga GR, et al. Lack of Transmission of Foot-andMouth Disease Virus From Persistently Infected Cattle to Naïve Cattle Under Field Conditions in Vietnam. Front Vet Sci. 2018;5:174.
6. Boggiatto PM, Buckley A, Cassmann ED, Seger H, Olsen SC, Palmer MV. Persistence of viral RNA in North American elk experimentally infected with an ancestral strain of severe acute respiratory syndrome coronavirus 2. Sci Rep. 2024 May 15;14:11116.
7. Conrady B, Garner G, Boklund A, Houe H, CalvoArtavia FF, Mortensen S, et al. Can the Danish veterinary authorities achieve their key performance indicator targets in the event of FMD disease outbreaks? Research Portal Denmark. 2024. Available from: https://local.forskningsportal.dk/local/dkicgi/ws/cris-link?id = ku-32cb6bfa-8cfd-450b-83622c8a28f2233a.
8. Conrady B, Garner G, Boklund A, Houe H, CalvoArtavia FF, Mortensen S, et al. Impact of external biosecurity measures on the spread of FMD in Denmark. Research Portal Denmark. 2024. Available from: https://local.forskningsportal.dk/local/dkicgi/ws/cris-link?id = ku-6dabab7d-f486-4c04-922b334ad225114c.
9. Eloiflin RJ, Grau-Roma L, Python S, Mehinagic K, Godel A, Libeau G, et al. Comparative pathogenesis of peste des petits ruminants virus strains of difference virulence. Vet Res. 2022;53:15.
10. Eschbaumer M, Stenfeldt C, Smoliga GR, Pacheco JM, Rodríguez LL, Li RW, et al. Transcriptomic Analysis of Persistent Infection with Foot-and-Mouth Disease Virus in Cattle Suggests Impairment of Apoptosis and Cell-Mediated Immunity in the Nasopharynx. PLoS One. 2016 Sep 14;11:e0162750.
11. Hägglund S, Valarcher JF. Persistent foot-and-mouth disease virus infection of the bovine nasopharynx is associated with suppression of innate and cellular immunity. Swedish University of Agricultural Sciences: Epsilon Open Archive; 2026.
12. Hamdy ME, Carlo MD, Hussein HA, Salah T, El-Deeb AH, Emara MM, et al. Development of gold nanoparticles biosensor for ultrasensitive diagnosis of foot and mouth disease virus. J Nanobiotechnology. 2018;16:48.
13. Henning A, Odendaal L, Loots AK, Quan M. Demonstrating persistence of foot-and-mouth disease virus in African buffalo using BaseScopeTM in situ hybridisation. Vet Res Commun. 2025;49.
14. Jena BR. Foot-and-Mouth Disease Carrier State in Livestock Population and its Diagnosis. J Anim Res. 2022 Dec;12:1-7.
15. Research Square [Preprint]. 2022. Available from: https://doi.org/10.21203/rs.3.rs-1834584/v1.
16. Kishimoto M, Tsuchiaka S, Rahpaya SS, Hasebe A, Otsu K, Sugimura S, et al. Development of a one-run real-time PCR detection system for pathogens associated with bovine respiratory disease complex. J Vet Med Sci. 2017;79:517-523.
17. Lakshmi IK, Putty K, Raut SS, Patil SR, Rao PP, Bhagyalakshmi B, et al. Standardization and application of real-time polymerase chain reaction for rapid detection of bluetongue virus. Vet World. 2018;11:452-458.
18. Liljander A, Sacchini F, Stoffel M, Schieck E, Stokar-Regenscheit N, Labroussaa F, et al. Reproduction of contagious caprine pleuropneumonia reveals the ability of convalescent sera to reduce hydrogen peroxide production in vitro. Vet Res. 2019;50:15.
19. Litz B, Pfaff F, Forth LF, Hägglund S, Valarcher JF, Beer M, et al. Persistent foot-and-mouth disease virus infection of the bovine nasopharynx is associated with suppression of innate and cellular immunity. PLoS One. 2026;21:e0340425.
20. Pacheco JM, Smoliga GR, O'Donnell V, Brito B, Stenfeldt C, Rodríguez LL, et al. Persistent Foot-andMouth Disease Virus Infection in the Nasopharynx of Cattle; Tissue-Specific Distribution and Local Cytokine Expression. PLoS One. 2015 May 11;10:e0125698.
21. Ríos L, Perera CL, Coronado L, Relova D, Álvarez A, Ganges L, et al. Multi-Target Strategy for Pan/Footand-Mouth Disease Virus Detection: A Combination of Sequences Analysis, in Silico Predictions and Laboratory Diagnostic Evaluation. Front Vet Sci. 2018;5:160.
22. Sarry M, Bernelin-Cottet C, Michaud C, Relmy A, Romey A, Salomez A, et al. Development of a primary cell model derived from porcine dorsal soft palate for foot-and-mouth disease virus research and diagnosis. Front Microbiol. 2023;14:1215347.
23. Schulz C, Fast C, Schlottau K, Hoffmann B, Beer M. Neglected Hosts of Small Ruminant Morbillivirus. Emerg Infect Dis. 2018;24:2334-2337.
24. Stenfeldt C, Arzt J. The Carrier Conundrum; A Review of Recent Advances and Persistent Gaps Regarding the Carrier State of Foot-and-Mouth Disease Virus. Pathogens. 2020 Mar 5;9:167.
25. Stenfeldt C, Eschbaumer M, Pacheco JM, Rekant SI, Rodríguez LL, Arzt J. Pathogenesis of Primary Footand-Mouth Disease Virus Infection in the Nasopharynx of Vaccinated and Non-Vaccinated Cattle. PLoS One. 2015 Nov 10;10:e0143666.
26. Stenfeldt C, Eschbaumer M, Rekant SI, Pacheco JM, Smoliga GR, Hartwig EJ, et al. The Foot-and-Mouth Disease Carrier State Divergence in Cattle. J Virol. 2016 Jun 24;90:6344-6364.
27. Stenfeldt C, Eschbaumer M, Smoliga GR, Rodríguez LL, Zhu J, Arzt J. Clearance of a persistent picornavirus infection is associated with enhanced pro-apoptotic and cellular immune responses. Sci Rep. 2017;7:18112.
28. Stenfeldt C, Pacheco JM, Singanallur NB, Vosloo W, Rodríguez LL, Arzt J. Virulence beneath the fleece; a tale of foot-and-mouth disease virus pathogenesis in sheep. PLoS One. 2019 Dec 19;14:e0227061.
29. Udahemuka JC, Aboge GO, Obiero G, Ingabire A, Beeton-Kempen N, Uwibambe E, et al. Investigation of foot and mouth disease virus and other animal pathogens in cattle, buffaloes and goats at the interface with Akagera National Park 2017-2020. BMC Vet Res. 2022;18:343.
30. Walker H, Bowman AS, Ferreira J, Nelson SW, Pairis-Garcia M, Arruda AG. Tonsil scrapings for porcine reproductive and respiratory syndrome virus detection in growing pigs under field conditions. J Swine Health Prod. 2021;29:72-80.
31. Zhu J, Stenfeldt C, Bishop E, Canter JA, Eschbaumer M, Rodríguez LL, et al. Mechanisms of Maintenance of Foot-and-Mouth Disease Virus Persistence Inferred From Genes Differentially Expressed in Nasopharyngeal Epithelia of Virus Carriers and Noncarriers. Front Vet Sci. 2020;7:340.