Chiesa, Francesco

Gili, S.

Nucera, Daniele Michele

Tursi, Massimiliano

English

Institutional Research Information System University of Turin

Diagnostic criteria of chronic lesionscaused by Taenia saginata metacestodesin bovine myocardium242F. CHIESA1, D. NUCERA2, S. GILI3, M. TURSI11 Dipartimento di Scienze Veterinarie, Università degli Studi di Torino, Via Leonardo da Vinci 44, 10095 Grugliasco, Torino, Italy2 Dipartimento di Scienze Agrarie, Forestali ed Alimentari, Università degli Studi di Torino, Via Leonardo da Vinci 44, 10095, Grugliasco, Torino, Italia3 Servizio Veterinario ASL TO1, Torino, ItaliaSUMMARYIntroduction - Humans are the only definitive host of the cestode Taenia saginata (beef tapeworm). The intermediate host ofthe larval form of T. saginata is mainly cattle. In cattle the eggs develop into the larval stage, Cysticercus bovis. Non-viable cy-sticerci undergoing degeneration can vary in appearance depending on the degree of inflammation, necrosis, and mineraliza-tion in the resulting lesion bovine cysticercosis can inflict serious economic losses to the cattle industry. These are mainly dueto condemnation, refrigeration and downgrading of infected carcasses. histological evaluation of T. saginata cysticerci suspectlesions is still considered the only laboratory diagnostic tool suitable for routine confirmation of undiagnosed lesions duringinspection procedures at slaughterhouse. There have been many studies to characterize the histopathology of cysticercosis,however none of them gave a practical and unbiased set of criteria in order to come to a sound based diagnosis.Materials and methods - 117 specimens of bovine ventricular myocardium positive for chronic lesions, presumptive of T. sa-ginata metacestodes at visual inspection at the abattoir, have been analyzed by histological examination and molecularmethods.Results and discussion - All the samples resulted positive for the presence of T.saginata Dna. Lymphocytes, granulocytes andnecrosis were the elements observed with higher frequency. In our study, histological observations of 9 out of 117 chronic le-sions found in bovine myocardium did not show typical cellularity of a parasitic granuloma. Statistical analysis demonstratethat the presence of lymphocytes and granulocytes is the most significant evidence of a chronic lesion left by the degenerationof the larval stage of the parasite, while a fibrotic capsule, though often present, shows a lower significance.Conclusions - The aim of this study was to demonstrate the practical use of a set of histological criteria for the identificationof chronic lesions caused by T. saginata metacestodes with the support of highly specific molecular methods.KEY WORDSTaenia saginata metacestodes, Bovine cysticercosis, Histopathology.INTRODUCTIONHumans are the only definitive host of the cestode Taeniasaginata (beef tapeworm). The intermediate host of the lar-val form (metacestode) of T. saginata is mainly cattle. Incattle the eggs develop into the larval stage, Cysticercus bo-vis. Bovine cysticercosis does not usually manifest itself cli-nically. In cattle the cysticerci degeneration takes place inseveral weeks and after one year many of them are dead orcalcified1. The life cycle is completed with humans inge-sting raw or inadequately cooked beef that contains viablecysticerci. T. saginata has a cosmopolitan distribution and is foundwidely in most countries that raise cattle2. bovine cysticer-cosis, although, according to meat inspecton data, in Euro-pe the prevalence ranges from 0.007% to 6.8%3,4, the realprevalence is considered to be at least ten times higher5,6.In contrast to T. solium, the clinical importance of T. sagina-ta is very limited. The presence of the tapeworm in the inte-stine can cause some abdominal discomfort, mild diarrhoeaand weight loss. In cattle, cysticercosis is usually subclinical.However, bovine cysticercosis can inflict serious economiclosses to the cattle industry7. These are mainly due to con-demnation, refrigeration and downgrading of infected car-casses. According to the Regulation (EC) no. 854/2004 all bo-vines of over 6 weeks of age have to be inspected for cysti-cercosis by incision and visual examination in masseter mu-scles and heart. If an animal has a generalised infestation, thecarcass and offal are declared unfit for human consumption.If the infestation is localised, however, the parts not infectedmay be declared fit for human consumption after having un-dergone a cold treatment.Although it appears that there are no true predilection sitesfor this parasite, those sites traditionally examined areamongst the best for detecting infection. Of these, the heartis preferred for detection based on the relatively high num-bers and frequencies with which cysticerci infect this organ,and the increased visibility of lesions due to the earlier dege-neration of cysticerci in cardiac muscle and concomitant in-Autore per la corrispondenza:Francesco Chiesa (francesco.chiesa@unito.it).F. Chiesa et al. Large Animal Review 2013; 19: 242-245 243flammatory response8. Non-viable cysticerci undergoing de-generation can vary in appearance depending on the degreeof inflammation, necrosis, and mineralization in the resul-ting lesion9. Cattle can harbor both viable (infective) and de-generate cysts concurrently5; thus recovery of only degenera-te cysts does not imply absence of infective cysts in the car-cass, or in herdmates. As well, viable cysts can persist in cat-tle for at least two to three years, and possibly for the life ofthe host10. Therefore, it is important to confirm cysticercosiseven in cases where suspect lesions are obviously degenera-ted and non-infective.Definitive diagnosis of degenerated lesions is often consi-dered difficult using gross, stereoscopic or histological exa-mination methods, on the grounds that the lesions no lon-ger contain identifiable parasite features, and are deemed tobe consistent histologically with chronic lesions of otheretiologies9.True indeed is the fact that in bovine cysticercosis, because ofthe absence of rostellar hooks in T. saginata cysticerci, andstanding the possibility that cysticerci in the heart and othertissues may not have developed fully prior to the onset of de-generation, the histological pattern available for the diagno-sis is singularly vague11.The onset of degeneration in mature cysts depends on theinflammatory response of the host but generally consists ofthe breakdown of the cyst wall and cuticle, followed by thedisappearance of following layers and eventually in the dis-solution of parasite remnants11.There have been many studies to characterize the histo-pathology of cysticercosis, some of which describe gradingcriteria based on characteristic histological features for bovi-ne or porcine cysticerci at various stages of degeneration11,12.However none of them gave a practical and unbiased set ofcriteria in order to come to a sound based diagnosis. In therecent past Polymerase chain reaction (PCR)-based methodshave been developed for the molecular identification of anumber of cestodes and larval stages thereof, among whichT. saginata1,9,13,14. The aim of this study was to demonstratethe practical use of a set of histological criteria for the iden-tification of chronic lesions caused by T. saginata metacesto-des with the support of molecular methods. A number of hi-stological elements, characteristic of chronic granulomatouslesions, have been taken in account and their presence hasbeen statistically associated with positivity to a specific T. sa-ginata PCR.MATERIALS AND METHODS117 specimens of bovine ventricular myocardium positivefor chronic lesions, presumptive of T. saginata metacestodesat visual inspection at the abattoir, were sampled for lightmicroscopic examination. Samples have been fixed in 10%buffered formalin for 24-48 hours, and processed for routineparaffin embedding. Sections of 3-µm thickness were stainedwith hematoxylin and eosin (HE).For each lesion, up to 25 mg of material was collected froma single excised cyst; effort was made to collect only the con-tent of the cyst, avoiding the cyst wall to reduce the amountof non target bovine DNA. DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany)was used for DNA extraction from all samples. Some minormodification in the protocols consisted of an overnight in-cubation with Proteinase K and a decrease in the volume ofthe final elution step (100 mL). Extracted DNA was quantified with a NanoDrop 2000spectrophotometer (ThermoScientific, Wilmington, NC).Figure 1 - Heart with multifocal whitish nodules at the level of theleft ventricular wall and the interventricular septum.Figure 2 - Particular of the heart wall with yellowish-white nodulepartially friable.Figure 3 - Histology of a parasitic granuloma in which it is possi-ble to detect a connective capsule and a mixed inflammatory infil-trate with giant cells and a central necrotic area. Hematoxylin andeosin stain, 10 X.PCR reactions were performed according to the protocolpublished by Chiesa et al (2010). Amplimers were resolvedby electrophoresis on a 2% agarose gel (Invitrogen), run inTris-acetate-ethylenediaminetetraacetic acid buffer for 70min at 110 V, and stained with ethidium bromide (0.4ng=mL) for 20 min.All statistical analyses were conducted using SAS software.Results were expressed as unadjusted odds ratios (OR) andcorresponding 95% confidence intervals and P values.RESULTSAll the samples resulted positive for the presence ofT.saginata Dna. Out of 117 samples, 9 did not demonstratethe presence of histological elements normally occurring inparasitic granuloma; PCR, however, was found to be positivefor 5 of them.Lymphocytes, granulocytes and necrosis were the elementsobserved with higher frequency, as shown in Table 1.In Table 2 are shown highly significant positive associationsfor each histological element and associations thereof ex-pressed as OR.The higher probability of the lesion being positive for T.saginata DNA presence has been in case of presence of gra-nulocytes: the probability that a chronic lesion found inthe bovine myocardium containing granulocytes would bepositive at the PCR is 16 folds higher than the probabilitythat such a lesion would yield a negative result at the mo-lecular test.DISCUSSIONSeveral different diagnostic tests have been described to sup-port visual diagnosis of T. saginata cysticercosis. Serologicalmethods, such as detecting circulating antigens or antibo-dies, both in live and slaughtered animals have been stu-died15,16. However, this technique failed to detect many lightinfections5, and the possibility of cross reaction with otherparasites indicates the need for further investigation17. Also,an immunohistochemical method has been proposed as apromising alternative by Ogunremi et al. (2004), but resultsshow that cross reaction with other taeniid species may oc-cur. Therefore, histological evaluation of T. saginata cysticer-ci suspect lesions is still considered the only laboratory dia-gnostic tool suitable for routine confirmation of undiagno-sed lesions during inspection procedures at slaughterhouse.The morphology and cellularity of these putative lesions canbe used to distinguish them from other commonly encoun-tered and somewhat similar lesions. Demonstration of a cy-sticercus- specific host structure is therefore necessary for adefinitive diagnosis11, but an accurate identification cannotalways be made, as such features are often not obvious in de-generated specimens and especially if the sample containscalcified tissues18.No standard criteria have been established for the histologi-cal evaluation of C. bovis-suspect lesions.In our study, histological observations of 9 out of 117 ch-ronic lesions found in bovine myocardium did not showtypical cellularity or only fibroblasts or capsule was pre-sent. 5 of those samples yield positive result to the PCR.This fact is explained by the localization of the histologicalexamination within the lesion: in order to test the same le-sion with both the histological and the molecular method,the sample was split before being formalin fixed. It may oc-cur that the part of the lesion undergoing to histologicalexamination would not be representative of the whole cel-lular population of the sample and that only the fibrous ca-psule was present.The presence of the capsule alone, though significantly asso-ciated with PCR positivity, is, somewhat surprising, less in-dicative of T. saginata metacestodes: presence of lymphocy-tes and granulocytes is a much more strong evidence of a ch-ronic lesion left by the degeneration of the larval stage of theparasite. Chronic lesions are the remnants of degenerated cy-sticerci undergone to host inflammatory response formonths and possibly many years; earlier degeneration of cy-sticerci in cardiac muscle and concomitant inflammatory re-sponse lead, with every probability, to an histological featu-res pattern dominated by those two inflammatory cells, whi-le macrophages, present in the earlier stage of the inflamma-tory response, tend to disappear with time.In conclusion we demonstrated that simple hematoxylin andeosin stain can be reliably used for the diagnosis of chroniclesions in bovine muscle suspected for T. saginata metace-stodes, even in case of lesions no longer containing identifia-ble parasite features, if histological patterns of parasitic gra-nuloma is observed. Even though the quality of the slidesproduced by frozen section is of lower quality than formalinfixed paraffin embedded tissue processing, the rapidity of thecryosection method would be of greater importance for thediagnosis of lesions in case of doubtful macroscopic inter-pretation at slaughterhouse.244 Diagnostic criteria of chronic lesions caused by Taenia saginata metacestodes in bovine myocardium*Highly significant.Tabella 1Histological elements Presence AbsenceLynphocytes 110 7Granulocytes 108 9Necrosis 97 20Macrophages 92 25Fibrous capsule 81 36Fibroblasts 33 84Table 2 - Odds Ratios for association between the presence of hi-stological elements and PCR positivity.Histological elements observed OR p valueLymphocites + granulocytes 16 0,002*Granulocytes 16 0,002*Lymphocites 13 0,01*Fib. capsule + granulocytes 9 0,005*Fib. capsule + necrosis 8 0,005*Fib.capsule+macrophages + lymphocites7 0,01*+ granulocytesFib. capsule + macrophages + lymphocites 7 0,01*Fib. capsule + macrophages 7 0,01*Fib. capsule 5 0,02F. Chiesa et al. Large Animal Review 2013; 19: 242-245 245References1. Geysen, D., Kanobana, K., Victor, B., Rodriguez-Hidalgo, R., De Borch-grave, J., Brandt, J., and Dorny, P. (2007) Validation of meat inspectionresults for Taenia saginata cysticercosis by PCR-restriction fragmentlength polymorphism, J Food Prot 70, 236-40.2. Murrell, K. D., and Dorny, P. (2005) WHO/FAO/OIE Guidelines for thesurveillance, prevention and control of taeniosis/cysticercosis, OIE.3. Cabaret, J., Geerts, S., Madeline, M., Ballandonne, C., and Barbier, D.(2002) The use of urban sewage sludge on pastures: the cysticercosis th-reat, Vet. Res 33, 575-597.4. Pawlowski, Z., and Schultz, M. G. (1972) Taeniasis and cysticercosis(Taenia saginata), Adv Parasitol 10, 269-343.5. Dorny, P., and Praet, N. (2007) Taenia saginata in Europe, VeterinaryParasitology 149, 22-24.6. Onyango-Abuje, J. A., Hughes, G., Opicha, M., Nginyi, K. M., Rugutt,M. K., Wright, S. H., and Harrison, L. J. S. (1996) Diagnosis of Taeniasaginata cysticercosis in Kenyan cattle by antibody and antigen ELISA,Veterinary Parasitology 61, 221-230.7. Yoder, D. R., Ebel, E. D., Hancock, D. D., and Combs, B. A. (1994) Epi-demiologic findings from an outbreak of cysticercosis in feedlot cattle,J Am Vet Med Assoc 205, 45-50.8. Scandrett, B., Parker, S., Forbes, L., Gajadhar, A., Dekumyoy, P., Waika-gul, J., and Haines, D. (2009) Distribution of Taenia saginata cysticerciin tissues of experimentally infected cattle, Veterinary Parasitology 164,223-231.9. Abuseir, S., Epe, C., Schnieder, T., Klein, G., and Kühne, M. (2006) Vi-sual diagnosis of Taenia saginata cysticercosis during meat inspection:is it unequivocal? Parasitology Research 99, 405-409.10. Urquhart, G. M., Armour, J., Duncan, J. L., Dunn, A. M., and Jennings,F. W. (1998) Veterinary parasitology.11. Silverman, P. H., and Hulland, T. J. (1961) Histological observations onbovine cysticercosis, Res. Vet. Sci 2, 248-252.12. Ogunremi, O., MacDonald, G., Geerts, S., and Brandt, J. (2004) Dia-gnosis of Taenia saginata cysticercosis by immunohistochemical test onformalin-fixed and paraffin-embedded bovine lesions, Journal of Vete-rinary Diagnostic Investigation 16, 438.13. Van der Logt, P. B., and Gottstein, B. (2000) Unidentified parasitic cy-sts in cattle, Vet Rec 146, 610-2.14. Chiesa, F., Dalmasso, A., Bellio, A., Martinetti, M., Gili, S., and Civera,T. (2010) Development of a Biomolecular Assay for Postmortem Dia-gnosis of Taenia saginata Cysticercosis, Foodborne Pathogens and Di-sease 405-409.15. Dorny, P., Vercammen, F., Brandt, J., Vansteenkiste, W., Berkvens, D.,and Geerts, S. (2000) Sero-epidemiological study of Taenia saginata cy-sticercosis in Belgian cattle, Veterinary Parasitology 88, 43-49.16. Van Kerckhoven, I., Vansteenkiste, W., Claes, M., Geerts, S., andBrandt, J. (1998) Improved detection of circulating antigen in cattleinfected with Taenia saginata metacestodes, Veterinary Parasitology76, 269-274.17. Brandt, J. R., Geerts, S., De Deken, R., Kumar, V., Ceulemans, F., Brijs,L., and Falla, N. (1992) A monoclonal antibody-based ELISA for thedetection of circulating excretory-secretory antigens in Taenia saginatacysticercosis, International Journal for Parasitology 22, 471.18. Schandevyl, P., and Vercruysse, J. (1982) Cysticercosis in cattle in Sene-gal, Veterinary Parasitology 11, 267.PROPOSTE EDITORIALI EV Per ordini e informazioni: E.V. Soc. Cons. a r.l. - Via Trecchi, 20 - 26100 Cremona Sezione Editoriale - Francesca Chiari - Enrico Soldi - Tel. 0372/403507 - 0372/403518 - Fax 0372/457091 - editoria@evsrl.it - www.evsrl.it/distribuzioneDI MARCO LO PRESTI et al.ATLANTE DI PATOLOGIA OVINA E CAPRINA1ª ed., 428 pagg., 1600 ill., GENOMA EDITORE, Settembre 2012Codice Articolo: LA171ISBN: 9788890426148Soci ass. fed. ANMVI € 128,00Non soci € 150,00CODIFAL’INFORMATORE FARMACEUTICO DI VETERINARIA E ZOOTECNIA23ª ed., 1551 pagg., EDRA, Luglio 2013Codice Articolo: PROF125ISBN: 9788821437489Soci ass. fed. ANMVI € 72,00Non soci € 85,00AA.VV. - In collaborazione con Farmadati ItaliaREPERTORIO DEI FARMACI VETERINARI - ANIMALI DA REDDITO ED EQUINI1ª ed., 800 pagg., Edizioni Veterinarie, Novembre 2011Codice Articolo: PROF115ISBN: 9788890426148Soci ass. fed. ANMVI € 45,00Non soci € 60,00

Diagnostic criteria of chronic lesions caused by Taenia saginata metacestodes in bovine myocardium

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Diagnostic criteria of chronic lesions caused by Taenia saginata metacestodes in bovine myocardium

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