Brucella melitensis in Iranian Dairy Cattle: Isolation, Molecular Identification, and Biotyping
-
Armin Kalantari
,
Hossein Esmaeili
,
Saeed Alamian
,
Seyed Mehdi Joghataei
,
Karim Amiri
,
Afshar Etemadi
Abstract
Background: Brucellosis, caused by Brucella species, remains a significant zoonotic and economic challenge in Iran, where Brucella melitensis is increasingly detected in dairy cattle due to close contact with infected sheep and goats. The persistence of B. melitensis highlights the limitations of existing control strategies and the need for improved approaches. Investigating B. melitensis in Iranian dairy cows is crucial due to their interaction with mixed sheep and goat populations.
Methods: In 2021, 73 lymphoid tissue samples were collected from reactor-positive cows in seven Iranian provinces. Samples were analyzed using traditional bacteriological methods, biotyping, and molecular tools such as AMOS-PCR and Bruce-ladder PCR for strain identification and characterization. Samples were collected from seven provinces in Iran, namely Tehran, Kerman, Isfahan, Alborz, Qazvin, West Azerbaijan, and Fars.
Results: Out of 73 lymph node samples collected from seven Iranian provinces, 15 (20.55%) tested positive for B. melitensis through culture and PCR. Culture results confirmed uniform growth conditions with no significant differences among provinces. Molecular analysis validated the identity of all isolates, with AMOS-PCR amplifying a 731 bp product and Bruce-ladder PCR confirming field strains through species-specific DNA fragments. Biotyping revealed that 14 isolates were B. melitensis Biotype 1, distributed across six provinces, while one isolate from Kerman Province was Biotype 3.
Conclusion: The prevalence of B. melitensis in Iranian dairy cattle highlights significant gaps in existing vaccination programs and the urgent need for cross-species protective vaccines. Given its zoonotic risks and public health impact, enhanced surveillance, advanced diagnostics, and region-specific strategies are critical for effective control.The RB51 vaccine has notable limitations, including its inability to protect against B. melitensis, lack of standardized protocols, and failure to aid in brucellosis eradication. Collaboration among public health authorities, veterinarians, and policymakers is critical to address these challenges and mitigate the spread of B. melitensis in Iran.
1. Esmaeili H. Brucellosis in Islamic republic of Iran. J Med Bacteriol 2015; 3(3-4):47-57.
2. Zange S, Scholz HC. Brucellosis. In: Sing A, editor. Zoonoses: Infections Affecting Humans and Animals. Cham: Springer International Publishing; 2023. p. 411-50.
3. Dadar M, Shahali Y, Fakhri Y. Brucellosis in Iranian livestock: A meta-epidemiological study. Microb Pathog 2021; 155:104921.
4. Rabbani Khorasgani M, Esmaeili H, Pourkarim MR, et al. Anti-brucella antibodies in blood donors in Boushehr, Iran. Comp Clin Path 2008; 17(4):267-9.
5. 5Sankhe S, Thorat V. Molecular characterization of Brucella melitensis detected in blood and serum samples of sheep and goats. Indian J Small Rumi 2023; 29(1):78-83.
6. Çelik E GSA, Büyük F, Otlu S, et al. Distribution of species and biotypes of Brucella isolates obtained from sheep and cattle abortions. Kafkas Univ Vet Fak Derg 2024; 30(4):549-57.
7. Mardani M (Shahid Beheshti University of Medical Sciences- MotNBC. The number of people suffering from Malata fever has increased / It is necessary to increase the allocation of funds to the country's veterinary organization Iran: Hakime-mehr; 2024 [Available from: https://hakimemehr.ir/000Kzo.
8. Esmaeili H, Tajik P, Ekhtiyarzadeh H, et al. Control and eradication program for bovine brucellosis in Iran: An epidemiological survey. J Vet Res 2012; 67(3):211-21.
9. Esmaeili H, Ekhtiyar zadeh H, Ebrahim zadeh H, et al. Evaluation of the national sheep and goat brucellosis control program in Iran. J Arak Univ Med Sci 2012; 14(7):9-20.
10. Nekuei Jahromi OA, Bahonar AR, Ekhtiarzadeh H, et al. Comparison of S19 and RB51 vaccines in control program of bovine brucellosis in the farms of Tehran province, before and after using of RB51 (1996-2006). Vet Res Bio Products 2009; 22(2):40-7.
11. Esmaeili H, Tajik P, Ekhtiyarzadeh H, et al. Control and eradication program for bovine brucellosis in Iran: an epidemiological survey. J Vet Res 2012; 67(3):211-21.
12. Sharifiyazdi H, Haghkhah M, Behroozikhah AM, et al. Bacteriological and molecular investigation of B. melitensis in dairy cows in Iran. Com Clin Path 2012; 21(3):269-73.
13. Zowghi E, Ebadi A, Yarahmadi M. Isolation and identification of Brucella organisms in Iran. Arch Clin Infect Dis 2008; 3(4):185-8.
14. Moriyón I, Blasco JM, Letesson JJ, et al. Brucellosis and One Health: Inherited and Future Challenges. Microorganisms 2023; 11(8).
15. Samartino LE, Enright FM. Pathogenesis of abortion of bovine brucellosis. Comparative Immunology, Microbiol Infect Dis 1993; 16(2):95-101.
16. Dadar M, Alamian S, Tadayon K, et al. Molecular characterization of zoonotic Brucella species isolated from animal and human samples in Iran. Acta Tropica 2022; 229:106363.
17. Dadar M, Alamian S. Isolation of Brucella melitensis from seronegative camel: potential implications in brucellosis control. Prev Vet Med 2020; 185:105194.
18. Qureshi KA, Parvez A, Fahmy NA, et al. Brucellosis: epidemiology, pathogenesis, diagnosis and treatment-a comprehensive review. Ann Med 2023; 55(2):2295398.
19. Esmaeili H, Almasi Chegeni S, Joghataei SM, et al. Etiology and risk factors of hemorrhagic abomasitis in goat kids. Sci Rep 2025; 15(1):6133.
20. WOAH. Brucellosis manual of diagnostic tests and vaccines for terrestrial animals. office international des epizooties, paris, france. paris: world organisation for animal health - office international des epizooties; 2016. p. 1092-106.
21. Al-Afifi AH, Alewy Almashhadany D, Al-Azazi ASH, et al. Prevalence of Brucella spp. in milk from aborted and non-aborted animals in Dhamar governorate, Yemen. Ital J Food Saf 2022; 11(4):10370.
22. Constable PD, Hinchcliff KW, Done SH, et al. Veterinary medicine: a textbook of the diseases of cattle, horses, sheep, pigs, and goats. 11 ed. United States: W.B. Saunders Ltd; 2017.
23. Markey B, Leonard F, Archambault M, et al. Clinical Veterinary Microbiology E-Book. 2nd ed ed. United Kingdom: Elsevier Health Sciences; 2013.
24. Ewalt DR, Bricker BJ. Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51. J Clin Microbiol 2000; 38(8):3085-6.
25. Bazrgari N, Garoosi GA, Dadar M. Genetic diversity and phylogenetic relationship of clinical isolates of brucella melitensis based on gene polymorphism of β subunit of RNA polymerase (rpoB) gene in Iran. Iran J Med Microbiol 2020; 14(5):425-40.
26. Dadar M, Alamian S, Behrozikhah AM, et al. Molecular identification of Brucella species and biovars associated with animal and human infection in Iran. Vet Res Forum 2019; 10(4):315-21.
27. López-Goñi I, García-Yoldi D, Marín CM, et al. Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains. J Clin Microbiol 2008; 46(10):3484-7.
28. Sharifiyazdi S, Kafi M, Haghkhah M, et al. Abortions in pregnant dairy cows after vaccination with Brucella abortus strain RB51. Vet Rec 2009; 165(19):570-1.
29. Di Giannatale E, De Massis F, Ancora M, et al. Typing of Brucella field strains isolated from livestock populations in Italy between 2001 and 2006. Vet Ital 2008; 44(2):383-8.
30. Di Giannatale E, De Massis F, Ancora M, et al. Typing of Brucella field strains isolated from livestock populations in Italy between 2001 and 2006. Vet Ital 2008; 44(2):383-8.
31. Álvarez J, Sáez JL, García N, et al. Management of an outbreak of brucellosis due to B. melitensis in dairy cattle in Spain. Res Vet Sci 2011; 90(2):208-11.
32. Sharma V, Pandey P, Singh P, et al. Host-pathogen interactions, diagnostics, and control measures for brucellosis in ruminants-a review. Indian J Anim Res 2023; 13(05):709-18.
33. Maadi MM, Haghi M. Prevalence of brucellosis in cattle in Urmia, Irán. Pakistan Veterinary Journal 2011; 31(1):81-2.
34. Verma DK. Brucellosis in animals and human beings with special reference to Indian subcontinent. International J Integrative sci Innov Tech 2013; 2:43-56.
35. Buyukcangaz E, Sen A. The first isolation of Brucella melitensis from bovine aborted fetus in Turkey. Journal of Biological and Environmental Sciences 2007; 1(3).
36. Wareth G, El-Diasty M, Abdel-Hamid NH, et al. Molecular characterization and antimicrobial susceptibility testing of clinical and non-clinical Brucella melitensis and Brucella abortus isolates from Egypt. One Health 2021; 13:100255.
37. Dal T, Kara SS, Cikman A, et al. Comparison of multiplex real-time polymerase chain reaction with serological tests and culture for diagnosing human brucellosis. J Infect Pub Health 2019; 12(3):337-42.
38. Demirpence M, Saytekin AM, Sareyyupoglu B, et al. Isolation and characterisation of Brucella melitensis by bacteriological and molecular methods from livestock in North Cyprus. Vet Med (Praha). 2022; 67(10):497-509.
39. Erami M, Momen-Heravi M, Razzaghi R, et al. The prevalence of Brucella biotypes isolated from sterile body fluids of patients with brucellosis in Kashan, Iran in 201. Avicenna J Clin Microbiol Infect 2016; 3(3):33516.
40. Moriyón I, Grilló MJ, Monreal D, et al. Rough vaccines in animal brucellosis: structural and genetic basis and present status. Vet Res 2004; 35(1):1-38.
41. Esmaeili H, Baghal Arani E, Mokhber Dezfouli M, et al. Study of the bacterial and nutritional causes of diarrhea in alpine and saanen kids. J Med Bacterio 2024; 12(1):1-8.
42. Blasco JM, Moreno E, Muñoz PM, et al. A review of three decades of use of the cattle brucellosis rough vaccine Brucella abortus RB51: myths and facts. BMC Vet Res 2023; 19(1):211.
43. Ragan VE. The animal and plant health inspection service (aphis) brucellosis eradication program in the United States. Vet Microbiol 2002; 90(1-4):11-8.
44. Olsen SC, Boggiatto PM. Characterization of the duration of immunity of Brucella abortus strain RB51 vaccination in cattle after experimental challenge. Prev Vet Med 2022; 206:105705.
45. Blasco JM, Moreno E, Moriyón I. Efficacy of Brucella abortus S19 and RB51 vaccine strains: A systematic review and meta-analysis. Transbound Emerg Dis 2022; 69(4):1670-3.
46. Ryskeldinova S, Zinina N, Kydyrbayev Z, et al. Registered influenza viral vector based Brucella abortus vaccine for cattle in Kazakhstan: Age-wise safety and efficacy studies. Front Cell Infect Microbiol 2021; 11:669196.
47. Esmaeili H, Ghorani M, Hamidiya Z, et al. Causes of abortion in Iranian goat herds and associated risk factors. Preventive Veterinary Medicine 2025; 234:106381.
48. Esmaeili H, Joghataei SM. Meningoencephalitic listeriosis in iranian sheep and goats. J Med Bacteriol 2024; 12(2):1-8.
49. Ullah I, Naz S, Khattak US, et al. Prevalence and associated risk factors of Brucella abortus and Brucella melitensis in humans and cattle populations: A comprehensive study. Com Immunol Microbiol Infect Dis 2024; 115:102276.
50. Olsen SC, Stoffregen WS. Essential role of vaccines in brucellosis control and eradication programs for livestock. Expert Rev Vaccines 2005; 4(6):915-28.
51. Esmaeili H, Ghorani M, Joghataei SM, et al. Live attenuated goatpox vaccination in pregnant Murcia-Granada goats: dosage implications and outcomes. BMC Vet Res 2024; 20(1):544.
2. Zange S, Scholz HC. Brucellosis. In: Sing A, editor. Zoonoses: Infections Affecting Humans and Animals. Cham: Springer International Publishing; 2023. p. 411-50.
3. Dadar M, Shahali Y, Fakhri Y. Brucellosis in Iranian livestock: A meta-epidemiological study. Microb Pathog 2021; 155:104921.
4. Rabbani Khorasgani M, Esmaeili H, Pourkarim MR, et al. Anti-brucella antibodies in blood donors in Boushehr, Iran. Comp Clin Path 2008; 17(4):267-9.
5. 5Sankhe S, Thorat V. Molecular characterization of Brucella melitensis detected in blood and serum samples of sheep and goats. Indian J Small Rumi 2023; 29(1):78-83.
6. Çelik E GSA, Büyük F, Otlu S, et al. Distribution of species and biotypes of Brucella isolates obtained from sheep and cattle abortions. Kafkas Univ Vet Fak Derg 2024; 30(4):549-57.
7. Mardani M (Shahid Beheshti University of Medical Sciences- MotNBC. The number of people suffering from Malata fever has increased / It is necessary to increase the allocation of funds to the country's veterinary organization Iran: Hakime-mehr; 2024 [Available from: https://hakimemehr.ir/000Kzo.
8. Esmaeili H, Tajik P, Ekhtiyarzadeh H, et al. Control and eradication program for bovine brucellosis in Iran: An epidemiological survey. J Vet Res 2012; 67(3):211-21.
9. Esmaeili H, Ekhtiyar zadeh H, Ebrahim zadeh H, et al. Evaluation of the national sheep and goat brucellosis control program in Iran. J Arak Univ Med Sci 2012; 14(7):9-20.
10. Nekuei Jahromi OA, Bahonar AR, Ekhtiarzadeh H, et al. Comparison of S19 and RB51 vaccines in control program of bovine brucellosis in the farms of Tehran province, before and after using of RB51 (1996-2006). Vet Res Bio Products 2009; 22(2):40-7.
11. Esmaeili H, Tajik P, Ekhtiyarzadeh H, et al. Control and eradication program for bovine brucellosis in Iran: an epidemiological survey. J Vet Res 2012; 67(3):211-21.
12. Sharifiyazdi H, Haghkhah M, Behroozikhah AM, et al. Bacteriological and molecular investigation of B. melitensis in dairy cows in Iran. Com Clin Path 2012; 21(3):269-73.
13. Zowghi E, Ebadi A, Yarahmadi M. Isolation and identification of Brucella organisms in Iran. Arch Clin Infect Dis 2008; 3(4):185-8.
14. Moriyón I, Blasco JM, Letesson JJ, et al. Brucellosis and One Health: Inherited and Future Challenges. Microorganisms 2023; 11(8).
15. Samartino LE, Enright FM. Pathogenesis of abortion of bovine brucellosis. Comparative Immunology, Microbiol Infect Dis 1993; 16(2):95-101.
16. Dadar M, Alamian S, Tadayon K, et al. Molecular characterization of zoonotic Brucella species isolated from animal and human samples in Iran. Acta Tropica 2022; 229:106363.
17. Dadar M, Alamian S. Isolation of Brucella melitensis from seronegative camel: potential implications in brucellosis control. Prev Vet Med 2020; 185:105194.
18. Qureshi KA, Parvez A, Fahmy NA, et al. Brucellosis: epidemiology, pathogenesis, diagnosis and treatment-a comprehensive review. Ann Med 2023; 55(2):2295398.
19. Esmaeili H, Almasi Chegeni S, Joghataei SM, et al. Etiology and risk factors of hemorrhagic abomasitis in goat kids. Sci Rep 2025; 15(1):6133.
20. WOAH. Brucellosis manual of diagnostic tests and vaccines for terrestrial animals. office international des epizooties, paris, france. paris: world organisation for animal health - office international des epizooties; 2016. p. 1092-106.
21. Al-Afifi AH, Alewy Almashhadany D, Al-Azazi ASH, et al. Prevalence of Brucella spp. in milk from aborted and non-aborted animals in Dhamar governorate, Yemen. Ital J Food Saf 2022; 11(4):10370.
22. Constable PD, Hinchcliff KW, Done SH, et al. Veterinary medicine: a textbook of the diseases of cattle, horses, sheep, pigs, and goats. 11 ed. United States: W.B. Saunders Ltd; 2017.
23. Markey B, Leonard F, Archambault M, et al. Clinical Veterinary Microbiology E-Book. 2nd ed ed. United Kingdom: Elsevier Health Sciences; 2013.
24. Ewalt DR, Bricker BJ. Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51. J Clin Microbiol 2000; 38(8):3085-6.
25. Bazrgari N, Garoosi GA, Dadar M. Genetic diversity and phylogenetic relationship of clinical isolates of brucella melitensis based on gene polymorphism of β subunit of RNA polymerase (rpoB) gene in Iran. Iran J Med Microbiol 2020; 14(5):425-40.
26. Dadar M, Alamian S, Behrozikhah AM, et al. Molecular identification of Brucella species and biovars associated with animal and human infection in Iran. Vet Res Forum 2019; 10(4):315-21.
27. López-Goñi I, García-Yoldi D, Marín CM, et al. Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains. J Clin Microbiol 2008; 46(10):3484-7.
28. Sharifiyazdi S, Kafi M, Haghkhah M, et al. Abortions in pregnant dairy cows after vaccination with Brucella abortus strain RB51. Vet Rec 2009; 165(19):570-1.
29. Di Giannatale E, De Massis F, Ancora M, et al. Typing of Brucella field strains isolated from livestock populations in Italy between 2001 and 2006. Vet Ital 2008; 44(2):383-8.
30. Di Giannatale E, De Massis F, Ancora M, et al. Typing of Brucella field strains isolated from livestock populations in Italy between 2001 and 2006. Vet Ital 2008; 44(2):383-8.
31. Álvarez J, Sáez JL, García N, et al. Management of an outbreak of brucellosis due to B. melitensis in dairy cattle in Spain. Res Vet Sci 2011; 90(2):208-11.
32. Sharma V, Pandey P, Singh P, et al. Host-pathogen interactions, diagnostics, and control measures for brucellosis in ruminants-a review. Indian J Anim Res 2023; 13(05):709-18.
33. Maadi MM, Haghi M. Prevalence of brucellosis in cattle in Urmia, Irán. Pakistan Veterinary Journal 2011; 31(1):81-2.
34. Verma DK. Brucellosis in animals and human beings with special reference to Indian subcontinent. International J Integrative sci Innov Tech 2013; 2:43-56.
35. Buyukcangaz E, Sen A. The first isolation of Brucella melitensis from bovine aborted fetus in Turkey. Journal of Biological and Environmental Sciences 2007; 1(3).
36. Wareth G, El-Diasty M, Abdel-Hamid NH, et al. Molecular characterization and antimicrobial susceptibility testing of clinical and non-clinical Brucella melitensis and Brucella abortus isolates from Egypt. One Health 2021; 13:100255.
37. Dal T, Kara SS, Cikman A, et al. Comparison of multiplex real-time polymerase chain reaction with serological tests and culture for diagnosing human brucellosis. J Infect Pub Health 2019; 12(3):337-42.
38. Demirpence M, Saytekin AM, Sareyyupoglu B, et al. Isolation and characterisation of Brucella melitensis by bacteriological and molecular methods from livestock in North Cyprus. Vet Med (Praha). 2022; 67(10):497-509.
39. Erami M, Momen-Heravi M, Razzaghi R, et al. The prevalence of Brucella biotypes isolated from sterile body fluids of patients with brucellosis in Kashan, Iran in 201. Avicenna J Clin Microbiol Infect 2016; 3(3):33516.
40. Moriyón I, Grilló MJ, Monreal D, et al. Rough vaccines in animal brucellosis: structural and genetic basis and present status. Vet Res 2004; 35(1):1-38.
41. Esmaeili H, Baghal Arani E, Mokhber Dezfouli M, et al. Study of the bacterial and nutritional causes of diarrhea in alpine and saanen kids. J Med Bacterio 2024; 12(1):1-8.
42. Blasco JM, Moreno E, Muñoz PM, et al. A review of three decades of use of the cattle brucellosis rough vaccine Brucella abortus RB51: myths and facts. BMC Vet Res 2023; 19(1):211.
43. Ragan VE. The animal and plant health inspection service (aphis) brucellosis eradication program in the United States. Vet Microbiol 2002; 90(1-4):11-8.
44. Olsen SC, Boggiatto PM. Characterization of the duration of immunity of Brucella abortus strain RB51 vaccination in cattle after experimental challenge. Prev Vet Med 2022; 206:105705.
45. Blasco JM, Moreno E, Moriyón I. Efficacy of Brucella abortus S19 and RB51 vaccine strains: A systematic review and meta-analysis. Transbound Emerg Dis 2022; 69(4):1670-3.
46. Ryskeldinova S, Zinina N, Kydyrbayev Z, et al. Registered influenza viral vector based Brucella abortus vaccine for cattle in Kazakhstan: Age-wise safety and efficacy studies. Front Cell Infect Microbiol 2021; 11:669196.
47. Esmaeili H, Ghorani M, Hamidiya Z, et al. Causes of abortion in Iranian goat herds and associated risk factors. Preventive Veterinary Medicine 2025; 234:106381.
48. Esmaeili H, Joghataei SM. Meningoencephalitic listeriosis in iranian sheep and goats. J Med Bacteriol 2024; 12(2):1-8.
49. Ullah I, Naz S, Khattak US, et al. Prevalence and associated risk factors of Brucella abortus and Brucella melitensis in humans and cattle populations: A comprehensive study. Com Immunol Microbiol Infect Dis 2024; 115:102276.
50. Olsen SC, Stoffregen WS. Essential role of vaccines in brucellosis control and eradication programs for livestock. Expert Rev Vaccines 2005; 4(6):915-28.
51. Esmaeili H, Ghorani M, Joghataei SM, et al. Live attenuated goatpox vaccination in pregnant Murcia-Granada goats: dosage implications and outcomes. BMC Vet Res 2024; 20(1):544.
| Issue | Vol 13 No 2 (2025) | |
| Section | Original Articles | |
| Keywords | ||
| AMOS-PCR Bruce-ladder PCR Brucella melitensis Brucellosis Dairy cattle Malta fever RB51 vaccine. | ||
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Kalantari A, Esmaeili H, Alamian S, Joghataei SM, Amiri K, Etemadi A. Brucella melitensis in Iranian Dairy Cattle: Isolation, Molecular Identification, and Biotyping. J Med Bacteriol. 2025;13(2):40-53.
