Investigating the Prevalence of Mycobacterium Avium Subspecies Paratuberculosis (MAP) in Industrial Dairy herds using Ziehl-Neelsen Staining, Culture, and PCR in Mashhad, Iran

Document Type : Research Articles


1 Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.

2 Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.


Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease in wild and domestic ruminants. Clinically, infected cattle show emaciation symptoms, diarrhea, and death. Asymptomatic subclinical cases can intermittently shed MAP through feces and milk and infect other cattle animals, thus increasing the risk of infection. The purpose of the present study was to detect the prevalence of this disease in dairy cattle. For this purpose, 348 samples were randomly collected from 15 cattle farms. All fecal samples were subjected to ZN staining and PCR of nucleotide sequences related to specific MAP gene fragments (IS900, F57) and were cultured in Harold's egg yolk culture medium after being disinfected with solution (0.0% HPC75). PCR testing of 116 fecal samples (33.3%, CI:95% 28.3-38.2), ZN staining of 23 samples (6.6%, CI: 95% 4-9.2), and fecal sample culture of only 15 samples (4.3%, CI:95% 2.3-6.3,) were infected with MAP. Comparison of test results showed poor agreement (kappa statistic: 0.19) between ZN staining and PCR results and poor agreement (kappa statistic: 0.13) between PCR test and fecal culture. This study highlights the advantages of PCR for detecting MAP in subclinically infected cattle compared to ZN staining and fecal culture. Therefore, it can be used for early detection and control of MAP in cattle and at-risk populations.


Main Subjects

1. Momotani E. Epidemiological situation and control strategies for paratuberculosis in Japan. Japanese Journal of Veterinary Research. 2012; 60:19-29. Doi: 10.14943/jjvr.60.suppl.s19.
2. Wadhwa A. Overview of Johne's disease immunology. Veterinary World. 2013; 6:901-904. Doi: 10.14202/vetworld.2013.901-904.
3. McAloon CG, Roche S, Ritter C, Barkema HW, Whyte P, More SJ et al. A review of paratuberculosis in dairy herds Part 1: Epidemiology. The Veterinary Journal. 2019; 246:59–65.
4. Ayele WY, Machackova M, Pavli I. The transmission and impact of paratuberculosis infection in domestic and wild ruminant. Veterinarni Medicina. 2001; 46(7-8): 205-224. Doi: 10.17221/7878-VETMED.
5. Raizman EA, Fetrow J, Wells SJ, Godden SM, Oakes MJ, Vazquez G. The association between Mycobacterium avium subsp. paratuberculosis fecal shedding or clinical Johne’s disease and lactation performance on two Minnesota, USA dairy farms. Preventive veterinary medicine. 2007; 78(3-4):179-195. Doi:10.1016/j.prevetmed.2006.10.006.
6. Rossi G, Grohn YT, Schukken YH, Smith RL. The effect of Mycobacterium avium ssp. paratuberculosis infection on clinical mastitis occurrence in dairy cows. Journal of dairy science. 2017; 100(9):7446-7454. Doi:org/10.3168/jds.2017-12721.
7. Lombard JE. Epidemiology and Economics of Paratuberculosis. Vet Clin Food Anim. 2011; 27:525- 535. Doi:10.1016/j.cvfa.2011.07.012.
8. Hassani M. Johne's disease in Iran: A systematic review and meta-analysis. Veterinary Researches and Biological Products. 2017; 30:139-147. Doi:10.22034/vj.2017.107807.
9. Waddell LA, Rajic A, Stark KDC, Mcewen SA. The zoonotic potential of Mycobacterium avium ssp. paratuberculosis: a systematic review and meta-analyses of the evidence. Epidemiology & Infection. 2015; 143:3135–3157. Doi:10.1017/S095026881500076X.
10. Chaubey KK, Singh SV, Gupta S, Singh M, Sohal JS, Kumar N et al. Mycobacterium avium subspecies paratuberculosis – an important food borne pathogen of high public health significance with special reference to India:an update. Veterinary Quarterly. 2017; 37(1):282-299.
11. Weigoldt M, Meens J, Doll  K, Fritsch I, Mobius P,Goethe R et al. Different proteome analysis of Mycobacterium avium subsp. paratuberculosis grown invitro and isolated from cases of clinical johnes disease. Microbiology. 2011; 157:557-565. Doi: 10.1099/mic.0.044859-0.
12. Park HT, Yoo HS. Development of vaccines to Mycobacterium avium subsp. paratuberculosis infection. Clinical and experimental vaccine research. 2016; 5(2):108-116.
13. Whittington RJ, Marsh I, McAllister S, Turner MJ, Marshall  D J, Fraser CA. Evaluation of Modified BACTEC 12B Radiometric Medium and Solid Media for Culture of Mycobacterium avium subsp. paratuberculosis from Sheep. Journal of clinical microbiology. 1999; 37(4):1077-1083. Doi: 10.1128/jcm.37.4.1077-1083.1999.
14. Douarre PE, Cashman W, Buckley J, Coffey A, Mahony JMO. Isolation and detection of Mycobacterium avium subsp. paratuberculosis (MAP) from cattle in Irland using both traditional culture and molecular based methods. Gut pathogens. 2010; 2:1-7. Doi:10.1186/1757-4749-2-11.
15. Pinedo PJ, Rae DO, Williams JE, Donovan GA, Melendez  P, Buergelt CD. Association among Results of Serum ELISA, Faecal Culture and Nested PCR on Milk, Blood and Faeces for the Detection of Paratuberculosis in Dairy Cows. Transboundary and Emerging Diseases. 2008; 55(2):125-133. Doi:10.1111/j.1865-1682.2007.01009.x.
16. Stabel JR. An improved method for cultivation of Mycobacterium paratuberculosis from bovine fecal samples and comparison to three other methods. Journal of Veterinary Diagnostic Investigation. 1997; 9(4):375-380. DOI: 10.1177/104063879700900406.
17. Singh AV, Singh SV, Singh PK, Sohal JS. Genotype diversity in Indian isolates of Mycobacterium avium subspecies paratuberculosis recovered from domestic and wild ruminants from different agro-climatic regions. Comparative Immunology, Microbiology and Infectious Diseases. 2010; 33:127-131. Doi:10.1016/j.cimid.2010.08.001.
18. Kupper J, Brandt H, Donat K, Erhardt G. Heritability estimates for Mycobacterium avium subspecies paratuberculosis status of German Holstein cows tested by fecal culture. Journal of dairy science. 2012; 95(5):2734-2739. Doi:org/ 10.3168/jds.2011-4994.
19. Mahmoodi Koohi P, Sadeghi-nasab A, Mohammadzadeh AM, Sharifi A, Bahari AA, Mosavari N. Identification of Mycobacterium avium subsp paratuberculosis infection in industrial dairy farms of Hamedan. Iranian Veterinary Journal. 2018;14(1):55-61.  Doi:10.22055/ivj.2017.67340.1832.
20. Zarei M, Ghorbanpour M, Tajbakhsh S, Mosavari N. Comparison of ELISA method, PCR and Ziehl-Neelsen staining of the rectal mucosa for detection of Mycobacterium avium subsp. paratuberculosis infection in cattle. Iranian Veterinary Journal. 2017;13(2):29-37. Doi:10.22055/ivj.2017.47784.1690.
21. Seyyedin M, Tadjbakhsh H, Salehi TZ. Identification of Mycobacterium avium subsp. paratuberculosis in fecal samples of Holstein-Friesian cattle using molecular and cultivation methods. Journal of Veterinary Research. 2010; 65(2):135-171.
22. Soltani M. Detection of Mycobacterium avium subsp. paratuberculosis in Kerman province's dairy cows using microbial culture, PCR and nested PCR methods. Iranian Journal of Animal Science Research. 2018; 10:263-273. Doi:10.22067/ijasr.v10i2.65920.
23. Grant IR, Rowe MT. Effect of chemical decontamination and refrigerated storage on the isolation of Mycobacterium avium subsp. paratuberculosis from heat-treated milk. Letters in applied microbiology. 2004; 38(4):  283-288. Doi:10.1111/j.1472-765X.2004.01498.x.
24. Vansnick E, De Rijk P, Vercammen F, Geysen D, Rigouts L, Portaels F. Newly developed primers for the detection of Mycobacterium avium subspecies paratuberculosis. Veterinary microbiology. 2004; 100(3-4):197-204. Doi:10.1016/j.vetmic.2004.02.006.
25. Ansari-Lari M, Haghkhah M, Bahramy A, Novin Baheran AM. Risk factors for Mycobacterium avium subspecies paratuberculosis in Fars province (Southern Iran) dairy herds. Tropical animal health and production. 2009; 41(4): 553–557. DOI 10.1007/s11250-008-9221-7.
26. Kaur P, Filia G, Singh SV, Patil PK, Kumar GR, Sandhu KS. Molecular epidemiology of Mycobacterium avium subspecies paratuberculosis: IS900 PCR identification and IS1311 polymorphism analysis from ruminants in the Punjab region of India. Comparative immunology, microbiology and infectious diseases. 2011; 34(2):163-169. Doi:10.1016/j.cimid.2010.09.002.
27. Nassiri M, Jahandar MH, Soltani M, Mahdavi M, Doosti M. Identification and strain determination of M. paratuberculosis (MAP) by PCR and REA methods based on IS900 and IS1311 insertion segments. Agricultural Biotechnology Journal. 2012; 4(1):83-96. Doi:10.22103/jab.2012.469.
28. Haghkhah M, Ansari-Lari M, Novin-Baheran AM, Bahramy A. Herd-level prevalence of Mycobacterium avium subspecies paratuberculosis by bulk-tank milk PCR in Fars province (southern Iran) dairy herds. Preventive veterinary medicine. 2008; 86(1-2):8-13. Doi:10.1016/j.prevetmed.2008.03.010.
29. Ebrahim Z, Tadayon K, Mosavari N. Genomic Characterization of the Vaccinal Strain of Mycobacterium avium Subspecies paratuberculosis (MAP) 316F by MIRU-VNTR. Medical Laboratory Journal. 2015; 9(4):15-21. Doi:10.18869/acadpub.mlj.9.4.15.
30. Sidoti F, Banche G, Astegiano S, Allizond V, Cuffini AM, Bergallo M. Validation and standardization of IS900 and F57 real-time quantitative PCR assays for the specific detection and quantification of Mycobacterium avium subsp. paratuberculosis. Canadian journal of microbiology. 2011; 57(5):347-354. Doi:10.1139/W11-022.
31. Ghaem Maghami S, Khosravi M, Ahmadi M, Denikoo A, Haghdin M, Koochakzadeh A. Study of the prevalence rate of john’s disease in markazi province and evaluation of absorbed elisa for adoption as a diagnostic method. Iranian Veterinary Journal. 2012; 8:54-9. 
32. Fernandez-Silva JA, Abdulmawjood A, Bulte M. Diagnosis and Molecular Characterization of Mycobacterium avium subsp. paratuberculosis from DairyCows in Colombia. Veterinary Medicine International. 2011:1-13. Doi:10.4061/2011/352561.
33. van Soolingen D, Haas PE, Kremer K. Restriction fragment Length polymorphism typing of Mycobacteria in Mycobacterium tuberculosis protocols. 2001:165-203. Doi: 10.1385/1-59259-147-7:165.
34. Ryan P, Bennett MW, Aarons  S, Lee G, Collins J K, O’Sullivan GC et al. PCR detection of Mycobacterium paratuberculosis in Crohn’s disease granulomas isolated by laser capture microdissection. Gut. 2002; 51:665-670. Doi: 10.1136/gut.51.5.665.
35. Tadayon K, Mosavari N, Keshavarz R, Shahmoradi A, Ghaderi R, Sekhavati M et al. Paratuberculosis in ruminants, a molecular search for traces of Mycobacterium avium subspecies paratuberculosis type I and type II in Iran. Veterinary Researches & Biological Products. 2016; 29(1):89-95. Doi: 10.22034/vj.2016.105749.
36. Thrusfield M. Diagnostic testing. :In Thrusfield M, Christley R, Brown H, Diggle PJ, French N, Howe K, et al. Veterinary epidemiology, 4 th End. Hoboken: John Wiley & Sons Ltd; 2018. 421-456.
Volume 15, Issue 2 - Serial Number 31
This issue XML file is being prepared.
July 2023
Pages 11-18
  • Receive Date: 25 January 2023
  • Revise Date: 01 March 2023
  • Accept Date: 09 May 2023
  • First Publish Date: 09 May 2023