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Detection of Mycobacterium avium subsp. paratuberculosis by several diagnostics techniques in clinical suspected sheep

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A total of thirty sheep with clinical symptomatology of paratuberculosis (Johne’s disease) were subjected to four diagnostic techniques: histopathological examination, bacteriological culture (in faeces and tissues), polymerase chain reaction (PCR) (in blood, tissue and faecal samples) and antibody responses (ELISA). Twenty-one (70.0%) animals showed histological lesions. Bacterial culture of both faeces and tissue revealed that 2 (6.7%) and 6 (20.0%) of the 30 sheep were infected, respectively. Mycobacterium avium subsp. 24 paratuberculosis (Map) was identified in 4 animals via PCR of faeces (13.3%), and in 19 (63.3%) by PCR in tissues. PCR in blood revealed 7 (23.3%) infected animals. Three (10.0%) animals showed antibodies against Map. A greater number of positive animals were detected by histopathological examination and PCR in tissues than by culture or ELISA. This study confirmed the clinical findings and results suggest that histopathology, PCR in tissues and in blood can improve the detection of Map in physically suspected animals and should be considered useful tools in the diagnosis of Map in suspected sheep.


sheep; histopathology; culture; PCR; ELISA; paratuberculosis

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Ayele WY, Bartos M, Svastova P, Pavlik I (2004) Distribution of Mycobacterium avium subsp. paratuberculosis in organs of naturally infected bull-calves and breeding bulls. Vet Microbiol 103:209-217

Bannantine JP, Wu CW, Hsu C, Zhou S, Schwartz DC, Bayles DO, Paustian ML, Alt DP (2012) Sreevatsan S, Kapur V & Talaat, A. M. Genome sequencing of ovine isolates of Mycobacterium avium subspecies paratuberculosis offers insights into host association. BMC Genomics 13:1

Bhide M, Chakurkar E, Tkacikova L, Barbuddhe S, Novak M, Mikula I (2006) IS900 PCR based detection and characterization of Mycobacterium avium subsp. paratuberculosis from buffy coat of cattle and sheep. Vet Microbiol 112:33-41

Clarke CJ, Little D (1996) The pathology of ovine paratuberculosis: gross and histological changes in the intestine and other tissues. J Comp Pathol 114:419-437

Coelho AC, Pinto ML, Silva S, Coelho AM, Rodrigues J, Juste RA (2007) Seroprevalence of ovine paratuberculosis infection in the Northeast of Portugal. Small Rum Res 71:298-303

Coelho AC, Pinto ML, Coelho AM, Rodrigues J (2008b) Coloração de Ziehl-Neelsen como método rápido de diagnóstico de paratuberculose ovina. Arq Bras Med Vet Zoo 60:1097-1102

Coelho AC, Pinto ML, Coelho AM, Rodrigues J, Juste R (2008) Estimation of the prevalence of Mycobacterium avium subsp. paratuberculosis by PCR in sheep blood. Small Rum Res 76:201-206

Garrido JM, Cortabarria N, Oguiza JA, Aduriz G, Juste RA (2000) Use of a PCR method on faecal samples for diagnosis of sheep paratuberculosis. Vet Microbiol 77:379-386

Garrido JM, Aduriz G, Juste RA, Geijo, MV (2000b). Los métodos de diagnóstico de la paratuberculosis en el ganado vacuno. Revista Bovis 93:49-61.

González J, Guijo MV, García-Pariente C, Verna A, Corpa JM, Reyes LE, Ferreras MC, Juste RA, García Marín JF, Pérez V (2005) Histopathological classification of lesions associated with natural paratuberculosis infection in cattle. J Comp Pathol 133:184-196

Gumber S, Eamens G, Whittington RJ (2006) Evaluation of a Pourquier ELISA kit in relation to agar gel immunodiffusion (AGID) test for assessment of the humoral immune response in sheep and goats with and without Mycobacterium paratuberculosis infection. Vet Microbiol 115:91-101

Gwózdz JM, Reichel MP, Murray A, Manktelow W, West DM, Thompson KG (1997) Detection of Mycobacterium avium subsp. paratuberculosis in ovine tissues and blood by the polymerase chain reaction. Vet Microbiol 51:233-244

Hilbink F, West DM, De Lisle GW, Kittelberger R, Hosie BD, Hutton J, Cooke MM, Penrose M. (1994). Comparison of a complement fixation test, a gel diffusion test and two absorbed and unabsorbed ELISAs for the diagnosis of paratuberculosis in sheep. Vet Micróbiol 41:107-116.

Hope AF, Kluver PF, Jones SL, Condron RJ (2000) Sensitivity and specificity of two serological tests for the detection of ovine paratuberculosis. Aus Vet J 78:850-856

Huntley JF, Whitlock RH, Bannantine JP, Stabel JR (2005) Comparison of diagnostic methods for Mycobacterium avium subsp. paratuberculosis in North American Bison. Vet Pathol 42:42-51

Kawaji S, Begg DJ, Plain KM, Whittington RJ (2011). A longitudinal study to evaluate the diagnostic potential of a direct faecal quantitative PCR test for Johne’s disease in sheep. Vet Microbiol 148:35-44.

Kumar V, Bhatia AK, Singh SV (2006) Evaluation of efficacy of the species specific antigens in the diagnosis of ovine and caprine Paratuberculosis using plate ELISA. J Immunol Immunopathol 8:48-53

Lavers CJ, Dohoo IR, McKenna SL, Keefe GP (2015) Sensitivity and specificity of repeated test results from a commercial milk enzyme-linked immunosorbent assay for detection of Mycobacterium avium subspecies paratuberculosis in dairy cattle. J Am Vet Med Assoc 246:236-244

Liapi M, Leontides L, Kostoulas P, Botsaris G, Iacovou Y, Rees C, Georgiu K, Smith GC, Naseby DC (2011). Bayesian estimation of the true prevalence of Mycobacterium avium subsp. paratuberculosis infection in Cypriot dairy sheep and goat flocks. Small Rumin Res 95:174-178.

Liapi M, Botsaris G, Slana I, Moravkova M, Babak V, Avraam M, Di Provvido A, Georgiadou S, Pavlik, . (2015). Mycobacterium avium subsp. paratuberculosis sheep strains isolated from Cyprus sheep and goats. Trans Emerg Dis 62:223-227.

Marsh I, Whittington R, Cousins D (1999) PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium based on polymorphisms in IS1311. Mol Cell Probes 13:115-126

McKenna SL, Keefe GP, Barkema HW, Sockett DC (2005) Evaluation of three ELISAs for Mycobacterium avium subsp. Paratuberculosis using tissue and faecal culture as comparison standards. Vet Microbiol 110:105-111

Mendes S, Boinas F, Albuquerque T, Fernandes L, Afonso A, Amado A (2004) Epidemiological studies on paratuberculosis in small ruminants in Portugal. Épidémiol Santé Anim 45:61-71

Miller J, Jenny AL, Payeur J (2002) Polymerase chain reaction detection of Mycobacterium tuberculosis complex and Mycobacterium avium organisms in formalin-fixed tissues from culture negative ruminants. Vet Microbiol 87:15-23

Moss MT, Sanderson JD, Tizard ML, Hermon-Taylor J, El-Zaatari FA, Markesich DC, Graham DY (1992) Polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp. silvaticum in long term cultures from Crohn’s disease and control tissues. Gut 33:1209-1213

Nielsen SS, Toft N (2008) Ante mortem diagnosis of paratuberculosis: a review of accuracies of ELISA, interferon-γ assay and faecal culture techniques. Vet Microbiol 129:217-235.

Pavlík I, Matlova L, Bartl J, Svastova P, Dvorska L, Whitlock R 2000 Parallel faecal and organ Mycobacterium avium subsp. paratuberculosis culture of different productivity types of cattle. Vet Microbiol 77:309-324

Pérez V, García-Marím JF, Badiola JJ (1996) Description and classification 385 of different types of lesion associated with natural paratuberculosis infection in sheep. J Comp Pathol 114:107-122

Reddacliff LA, Vadali A, Whittington RJ (2003) The effect of decontamination protocols on the numbers of sheep strain Mycobacterium avium subsp. paratuberculosis isolated from tissues and faeces. Vet Microbiol 95:271-282

Sevilla I, Singh SV, Garrido JM, Aduriz G, Rodríguez S, Geijo MV, Whittington RJ, Saunders V, Whitlock RH, Juste RA (2005) PCRREA genotype of paratuberculosis strains isolated from different host species and geographical locations. Sci Tech Rev 24:1061-1066

Silva, K., Begg, D., Carter, N., Taylor, D., Di Fiore, L., & Whittington, R. (2010). The early lymphocyte proliferation response in sheep exposed to Mycobacterium avium subsp. Paratuberculosis compared to infection status. Immunobiology, 215(1), 12-25.

Singh AV, Singh SV, Sohal JS, Singh PK (2009) Comparative potential of modified indigenous, indigenous and commercial ELISA kits for diagnosis of Mycobacterium avium subspecies paratuberculosis in goat and sheep. Indian J Exp Biol 47:379-382

Singh SV, Singh PK, Gupta S, Chaubey KK, Singh B, Kumar A, Singh V, Kumar N (2013) Comparison of microscopy and blood-PCR for the diagnosis of clinical Johne’s disease in domestic ruminants. Iranian J Vet Res 14:345-349

Singh V, Singh PK, Singh AV, Sohal JS, Kumar N, Chaubey KK, Gupta S, Rawat KD, Kumar A, Bhatia AK, Srivastav AK, Dhama K (2014) ‘Bio-Load’ and bio-type profiles of Mycobacterium avium subspecies paratuberculosis infection in the domestic livestock population endemic for Johne’s disease: a survey of 28 years (1985–2013) in India. Trans Emerg Dis 61:43-55

Stabel JR (1997). An improved method for cultivation of Mycobacterium paratuberculosis from bovine faecal samples and comparison to three other methods. J Vet Diagnos Invest 9:375-380.

Verdugo C, Jones G, Johnson W, Wilson P, Stringer L, Heuer C (2014) Estimation of flock/herd-level true Mycobacterium avium subspecies paratuberculosis prevalence on sheep, beef cattle and deer farms in New Zealand using a novel Bayesian model. Prev Vet Med 117:447-455

Whitlock RH, Wells SJ, Sweeney RW, Van Tiem J (2000) ELISA and faecal culture for paratuberculosis (Johne’s disease): sensitivity and specificity of each method. Vet microbial 77:387-398.

Whittington RJ, Marsh I, McAllister S, Turner MJ, Marshall DJ, Fraser, CA (1999). Evaluation of Modified BACTEC 12B Radiometric Medium and Solid Media for Culture of Mycobacterium aviumsubsp. paratuberculosis from Sheep. Journal of clinical microbiology, 37, 1077-1083.

Whittington RJ, Marsh IB, Whitlock RH (2001) Typing of IS1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with a strain of Mycobacterium avium subsp. paratuberculosis distinct from that occurring in cattle and other domesticated livestock. Mol Cell Probes 15:139-145

Zimmer K, Drager KG, Klawonn K, Hess RG (1999). Contribution to the Diagnosis of Johne’s Disease in Cattle. Comparative Studies on the Validity of Ziehl-Neelsen Staining, Faecal Culture and a Commercially Available DNA-Probe® Test in Detecting Mycobacterium paratuberculosis in Faeces from Cattle. J Vet Med Series B, 46:137-140


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