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Clinical and molecular investigation of johne’s disease among small ruminants in Makkah, Saudi Arabia

Atif H. Asghar, Ibrahim HA Abd El-Rahim, Amr M Mohamed, Omar B Ahmed*


Paratuberculosis (PTB) or Johne’s disease (JD) is a chronic debilitating disease caused by the Mycobacterium avium subsp. paratuberculosis (MAP). JD affects a wide range of animals including ruminants and characterized by enteritis and progressive diarrhea. The current study aimed to implement molecular tools for detection and identification of MAP among clinically suspected small ruminants in Makkah region, Kingdom of Saudi Arabia (KSA). A total of 2660 small ruminants from five different farms around Makkah were clinically investigated for characteristic signs of JD during the period of November 2013 to February of 2014. Out of investigated animals, 16 cases were selected as being suspected of JD infection based on the associated clinical symptoms (emaciation and unthriftiness with or without persistent diarrhea). Rectal scrapings were collected from all suspected animals and were subjected for molecular examination. Three different genetic targets were evaluated including 16S rDNA, insertion sequence 900 (IS900) and intergenic spacer (IGS). Initial examination of suspected specimens by amplification of universal bacterial primers for 16S rDNA revealed positive results in 6 out of 16 cases (37.5%). Further amplification of the Mycobacterium-specific IGS target from 16s rDNA-positive samples revealed the detection of 4 Mycobacterium species. Sequence analysis of the IGS sequence of these 4 Mycobacterium species revealed 2 cases only as Mycobacterium avium paratuberculosis. This finding was confirmed by the detection of MAP-specific IS900 target only from the same 2 cases. In conclusion, the current study genetically documented the first report of MAP (the causative agent of JD) among small ruminants of Makkah region.


Johne’s disease; microscopic examination; molecular investigation; small ruminants; Saudi Arabia.

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Clarke CJ, The pathology and patho- genesis of paratuberculosis in ruminants and other species, Journal of Comparative Pathology, 1997, 116: 217–261.

Richardson EKB, More SJ, Direct and indirect effects of Johne's disease on farm and animal productivity in an Irish dairy herd, Ir Vet J, 62, 2009:526–532.

Collins MT, Clinical approach to control of bovine paratuberculosis. J Am Vet Med Assoc, 1994, 204: 208-210.

Sweeney RW, Whitlock RH, Buckley CL, Evaluation of a commercial enzyme-linked immunosorbent assay for the diagnosis of paratuberculosis in dairy cattle. J. Vet. Diagn. Invest, 1995, 7: 488- 493.

Whitlock RH, Buergelt C, Preclinical and clinical manifestations of paratuberculosis (including pathology). Vet Clin North Am Food Anim Pract,1996, 12: 345–356.

Tiwari A, VanLeeuwen JA, McKenna SLB, et al.: 2006, Johne’s disease in Canada: Part I: Clinical symptoms, pathophysiology, diagnosis, and prevalence in dairy herds. Can Vet J, 2006, 47: 874–882.

De Lisle GW, Duncan JR: 1981, Bovine paratuberculosis III. An evaluation of a whole blood lymphocyte transformation test. Can J Comp Med, 1981, 45: 304–309.

Tiwari A, Vanleeuwen JA, Dohoo IR, Stryhn H, Keefe GP, Effects of seropositivity for bovine leukemia virus, Mycobacterium avium subspecies paratuberculosis, and Neospora caninum on calving to conception interval in maritime Canadian dairy cattle. Proc Soc Vet Epidemiol Prev Med, Warwick, England, 2003, 243–252.

Tiwari A1, VanLeeuwen JA, Dohoo IR, Stryhn H, Keefe GP, Haddad JP, Effects of seropositivity for bovine leukemia virus, bovine viral diarrhoea virus, Mycobacterium avium subspecies paratuberculosis, and Neospora caninum on culling in dairy cattle in four Canadian provinces, Vet Microbiol, 2005, 109: 147–158.

Merkal RS, Thurston JR, Comparison of Mycobacterium paratuberculosis and other mycobacteria, using standard cytochemical tests. Am J Vet Res, 1996, 27: 519–521.

Kennedy DJ, Benedictus G, Control of Mycobacterium avium subsp. paratuberculosis infection in agricultural species. Rev Sci Tech, 2001, 20: 151–179.

Lepper AW, Wilks CR, Kotiw M, Sequential bacteriological observations in relation to cell-mediated and humoral antibody responses of cattle infected with Mycobacterium paratuberculosis and maintained on normal or high iron intake. Aust Vet J, 1989, 66: 50–55.

Kalis CH, Collins MT, Barkema HW, Hesselink JW, Certification of herds as free of Mycobacterium paratuberculosis infection: actual pooled faecal results versus certification model predictions. Prev Vet Med, 2004, 65: 189–204.

McKenna SL, Keefe GP, Barkema HW, McClure J, VanLeeuwen J A, Hanna P, and Sockett D C, Cow-level prevalence of paratuberculosis in culled dairy cows in Atlantic Canada and Maine, J Dairy Sci, 2004, 87: 3770–3777. S. L. B.

Dargatz DA, Byrum BA, Barber LK, Sweeney RW, Whitlock RH, Shulaw WP, Jacobson RH, Stabel JR, Evaluation of a commercial ELISA for diagnosis of paratuberculosis in cattle, J Am Vet Med Assoc, 2001, 218: 1163–1166.

Milner AR1, Mack WN, Coates KJ, Hill J, Gill I, Sheldrick P., The sensitivity and specificity of a modified ELISA for the diagnosis of Johne’s disease from a field trial in cattle, Vet Microbiol, 1990, 25: 193–198.

Stich RW1, Byrum B, Love B, Theus N, Barber L, Shulaw WP, Evaluation of an automated system for non-radiometric detection of Mycobacterium avium paratuberculosis in bovine feces. J Microbiol Methods, 2004, 56: 267–275.

Douarre PE, Cashman W, Buckley J, Coffey A and O'Mahony J M , Isolation and detection of Mycobacterium avium subsp. paratuberculosis (MAP) from cattle in Ireland using both traditional culture and molecular based methods, Gut Pathogens, 2010, 2: 11.

Springer B1, Stockman L, Teschner K, Roberts GD, Böttger EC., Two- laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods. J Clin Microbiol, 1996, 34: 296–303.

Kim BJ, Lee SH, Lyu MA: 1999, Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB). J Clin Microbiol, 1999, 37: 1714–1720.

Moss MT, Sanderson JD, Tizard MLV, et al.: 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, 1992, 33: 1209–1213.

Al-Dughaym, AM, Fadl El mula A, Mohamed GE, et al, First report of an outbreak of ovine septicaemic listeriosis in Saudi Arabia, Rev sci tech Offint Epiz, 2001, 20: 777-783.

Al-Naeem EME, Abu Elzein, Al-Afale AI, Epizootiological aspects of peste des petits ruminants and rinderpest in sheep and goats in Saudi Arabia. Rev. sci. tech. Off. int. Epiz,2000, 19: 855-858.

Alhebabi AM, Alluwaimi AM, Paratuberculosis in Camel (Camelus dromedarius): The Diagnostic Efficiency of ELISA and PCR. The Open Veterinary Science Journal, 2010, 4: 41-44.

Al Hajri SM, Alluwaimi AM, ELISA and PCR for evaluation of subclinical paratuberculosis in the Saudi dairy herds, Vet Microbiol, 2007, 121: 384-385.

Al-dubaib MA, Mahmoud OM, Paratuberculosis of goats at Qassim region of central saudi arabia. Bulgarian Journal of Veterinary Medicine, 2008, 11: 65−69.

Alluwaimi AM, Hatem ME, Almousa JM, The efficacy of gel immunodiffusion and fecal smear tests for diagnosis of ovine paratuberculosis in sheep in Saudi Arabia, Egyp J Immunol, 1999, 7: 29-32.

Manning EJ, Collins MT, Mycobacterium avium subsp. paratuberculosis: pathogen, pathogenesis and diagnosis, Rev Sci Tech, 2001, 20: 133–150.

Quinn PJ., Markey BK, Carter ME, Donnelly W J, Leonard F C, Mycobacterium species In: Veterinary microbiology and microbial diseases. 1st ed, Iowa State University Press Blackwell Science 1994.

Bollet C, Gevaudan MJ, de Lamballerie X, de, Zandotti C, Micco P de, A simple method for the isolation of chromosomal DNA from Gram positive or acid-fast bacteria, Nucleic Acids Res,1991, 19: 1955.

Hall L, Doerr KA, Wohlfiel SL, Roberts GD, Evaluation of the MicroSeq system for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory. J Clin Microbiol, 2003, 41: 1447–1453.

Mohamed AM1, Kuyper DJ, Iwen PC, Ali HH, Bastola DR, Hinrichs SH, Computational approach involving use of the internal transcribed spacer-1 region for identification of Mycobacterium species. J Clin Microbiol, 2005, 43: 3811–3817.

Khare S, Ficht TA, Santos RL, Romano J, Ficht AR, Zhang S, Grant IR, Libal M, Hunter D, Adams LG., Rapid and sensitive detection of Mycobacterium avium subsp. paratuberculosis in bovine milk and feces by a combination of immunomagnetic bead separation-conventional PCR and real-time PCR. J Clin Microbiol, 2004, 42: 1075-1081.

Green EP, Tizard ML, Moss MT, Thompson J, Winterbourne DJ, McFadden JJ and Hermon-Taylor J, Sequence and characteristics of IS900, an insertion element identified in a human Crohn’s disease isolate of Mycobacterium paratuberculosis, Nucleic Acids Res,1989, 17: 9063–9073.

Fidler HM, Thurrell W, Johnson NM, Rook GA, McFadden JJ, Specific detection of Mycobacterium paratuberculosis DNA associated with granulomatous tissue in Crohn’s disease, Gut, 1994, 35: 506–510.

Mishina D, Katsel P, Brown ST, Gilberts E C , and Greenstein R J ., On the etiology of Crohn disease, Proc Natl Acad Sci USA,1996, 93: 9816–9820.

Sanderson JD, Moss MT, Tizard MLV, et al.: 1992, Mycobacterium paratuberculosis DNA in Crohn’s disease tissue, Gut, 1992, 33: 890–896.

Uzoigwe JC, Khaitsa ML, Gibbs PS: 2007, Epidemiological evidence for Mycobacterium avium subspecies paratuberculosis as a cause of Crohn's Disease, Epidemiology and Infection,2007, 135: 1057-1068.

Eaton SL, Rocchi M, González L, Hamilton S, Finlayson J, Sales J, Jeffrey M, Steele PJ, Dagleish MP, Rodger SM, Reid HW, Chianini F, Immunological differences between susceptible and resistant sheep during the preclinical phase of scrapie infection, J Gen Virol, 2007, 88: 1384-1391.

Collins MT, Update on paratuberculosis: 1. Epidemiology of Johne's disease and the biology of Mycobacterium paratubertulosis, Irish Vet J, 2003, 56: 565-574.

Roussel AJ, Fosgate GT, Manning EJB, Collins MT, Association of fecal shedding of mycobacteria with high ELISA-determined seroprevalence for paratuberculosis in beef herds, J Am Vet Med Assoc, 2007, 230: 890-895.

Cloud JL, Neal H, Rosenberry R, C Y Turenne, M Jama, DR. Hillyard, and K. C. Carroll, Identification of Mycobacterium spp. by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries, J Clin Microbiol, 2002, 40:400–406.

Han XY, Pham AS, Tarrand JJ, Sood PK, Rapid and Accurate Identification of Mycobacteria by Sequencing Hypervariable Regions of the 16S Ribosomal RNA Gene, Am J Clin Pathol, 2002, 118: 796-801.

Mohamed AM, Abou El-Ella GA, Nasr EA, Phenotypic and molecular typing of tuberculous and nontuberculous Mycobacterium species from slaughtered pigs, Egypt J Vet Diagn Invest, 2009, 21: 48–52

Mohamed AM, Iwen PC, Tarantolo S, Hinrichs SH, Mycobacterium nebraskiae sp. nov., a new slow-growing scotochromogenic Mycobacterium, Int J Syst Evol Microbiol, 2004, 54: 2057–2060.

Roth A1, Fischer M, Hamid ME, Michalke S, Ludwig W, Mauch H. Differentiation of phylogenetically related slowly growing mycobacteria based on 16S-23S rRNA gene internal transcribed spacer sequences, J Clin Microbiol, 1998, 36: 139–147.

Whipple DL, Le Febvre RB, Andrews RE, and Thiermann A B, Isolation and analysis of restriction endonuclease digestive patterns of chromosomal DNA from Mycobacterium paratuberculosis and other Mycobacterium species, J Clin Microbiol, 1987, 25: 1511–1515.



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