(ERRATA) A Comparison of Bacteriological Culture, Serology, and Quantitative PCR for Detecting Brucellosis in Ewes with a History of Abortion

Iranian Journal of Veterinary Science and Technology

دوره 17، شماره 3 - شماره پیاپی 40، آذر 2025، صفحه 74-75   

نوع مقاله: Erratum

شناسه دیجیتال (DOI): 10.22067/ijvst.2025.47340

نویسندگان

Mohammad Javad Aminzadeh¹؛ Hamideh Kalateh Rahmani²؛ Khadijeh Hashemii³؛ Narges Khaleghnia⁴؛ Mohammad Azizzadeh¹؛ Pezhman Mirshokraei⁵

¹Department of Clinical Sciences, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.

²Department of Pathobiology, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.

³Division of Biotechnology, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran & Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.

⁴Centre of Excellence in Ruminant Abortion and Neonatal Mortality, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.

⁵Department of Clinical Sciences, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran & Centre of Excellence in Ruminant Abortion and Neonatal Mortality, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.

چکیده

The zoonotic disease brucellosis is a serious public health and livestock industry concern. In the present study, we used bacteriological culture, RBT, and qPCR to determine the prevalence of brucellosis in the serum and milk samples of sheep with a history of abortion. Serum and milk samples were obtained from 100 sheep aged 3-5 years. In order to determine the prevalence of brucellosis, a modified RBT was performed on serum samples, Brucella was isolated from milk by bacteriological culture, and qPCR was applied to detect bacterial DNA in milk. The prevalence of brucellosis using modified RBT, bacteriological culture, and qPCR was 32%, 42%, and 44%, respectively. By considering qPCR as the standard, modified RBT showed a sensitivity of 95%, a specificity of 100%, an accuracy of 98%, a PV+ of 100%, and a PV- of 97%. The sensitivity, specificity, accuracy, PV+, and PV- for bacteriological culture were 77%, 100%, 90%, 100%, and 85%, respectively. The agreement between qPCR and modified RBT was 0.959 (95% CI: 0.896-1), between qPCR and bacteriological culture was 0.792 (95% CI: 0.667-0.897), and between modified RBT and bacteriological culture was 0.831 (95% CI: 0.709-0.38). Based on the results, bacterial isolation from sheep milk is not recommended except in specific cases due to its low sensitivity, as well as its time-consuming and hazardous nature. However, the modified RBT can be used as a routine method because of its cost-effectiveness, higher sensitivity, and higher accuracy compared to bacterial isolation. Moreover, qPCR is recommended as the gold standard test for detecting brucellosis in sheep milk, especially in those with a history of abortion.

کلیدواژه‌ها

Brucellosis؛ Modified Rose Bengal؛ qPCR؛ Sheep

مراجع

1.    Cardoso PG, Macedo GC, Azevedo V, Oliveira SC. Brucella spp noncanonical LPS: structure, biosynthesis, and interaction with host immune system. Microbial cell factories. 2006;5(1): 1-11.doi: 10.1186/1475-2859-5-13
2.    Ilhan Z, Boynukara B, Solmaz H, Ekin I, Aksakal A, Gulhan T. Detection of Brucella melitensis DNA in the milk of sheep after abortion by PCR assay. Archivos de medicina veterinaria. 2008;40(2):141-6. doi: 10.4067/S0301-732X2008000200005
3.    Zowghi E, Ebadi A, Yarahmadi M. Isolation and identification of Brucella organisms in Iran. Archives of Clinical Infectious Diseases. 2008;3(4):185-8. doi: f5a67937da41b22526b49ea55a9b976330913693
4.    Kaya O, Akçam FZ, Yayli G. Investigation of the in vitro activities of various antibiotics against Brucella melitensis strains. Turkish Journal of Medical Sciences. 2012;42(1):145-8. doi: 10.3906/sag-1009-1129.
5.    Khurana SK, Sehrawat A, Tiwari R, Prasad M, Gulati B, Shabbir MZ, et al. Bovine brucellosis–a comprehensive review. Veterinary Quarterly. 2021;41(1):61-88. doi: 10.1080/01652176.2020.1868616.
6.    Gupta V, Verma DK, Rout P, Singh S, Vihan V. Polymerase chain reaction (PCR) for detection of Brucella melitensis in goat milk. Small Ruminant Research. 2006;65(1-2):79-84.Doi: 10.1016/j.smallrumres.2005.05.024
7.    Benkirane A. Ovine and caprine brucellosis: World distribution and control/eradication strategies in West Asia/North Africa region. Small ruminant research. 2006;62(1-2):19-25.doi: 10.1016/j.smallrumres.2005.07.032. 
8.    Seleem MN, Boyle SM, Sriranganathan N. Brucellosis: a re-emerging zoonosis. Veterinary microbiology. 2010;140(3-4):392-8. doi: 10.1016/j.vetmic.2009.06.021
9.    Whatmore AM, Koylass MS, Muchowski J, Edwards-Smallbone J, Gopaul KK, Perrett LL. Extended multilocus sequence analysis to describe the global population structure of the genus Brucella: phylogeography and relationship to biovars. Frontiers in microbiology. 2016;7:2049. doi: 10.3389/fmicb.2016.02049
10.    Behroozikhah AM, Bagheri Nejad R, Amiri K, Bahonar AR. Identification at biovar level of Brucella isolates causing abortion in small ruminants of Iran. Journal of pathogens. 2012;2012. doi: 10.1155/2012/357235.
11.    Zambriski JA, Maves RC, Nydam DV, Ayvar V, Cepeda D, Castillo R, et al. Effect of storage temperature and sample volume on Brucella melitensis isolation from goat milk. International Journal of Tropical Disease and Health. 2012;2012:207-13. doi: 10.9734/IJTDH/2012/1738.
12.    Pandya A, Ghodke K. Goat and sheep milk products other than cheeses and yoghurt. Small Ruminant Research. 2007;68(1-2):193-206.Doi: 10.1016/j.smallrumres.2006.09.007
13.    YAEGER MJ, HOLLER LD. Bacterial causes of bovine infertility and abortion. Current therapy in large animal theriogenology: Elsevier; 2007. 389-99.
14.    Di Bonaventura G, Angeletti S, Ianni A, Petitti T, Gherardi G. Microbiological laboratory diagnosis of human brucellosis: An overview. Pathogens. 2021;10(12):1623. doi: 10.3390/pathogens10121623. 
15.    Rossetti CA, Arenas-Gamboa AM, Maurizio E. Caprine brucellosis: A historically neglected disease with significant impact on public health. PLoS neglected tropical diseases. 2017;11(8):e0005692. doi: 10.1371/journal.pntd.0005692.
16.    Lindahl-Rajala E, Hoffman T, Fretin D, Godfroid J, Sattorov N, Boqvist S, et al. Detection and characterization of Brucella spp. in bovine milk in small-scale urban and peri-urban farming in Tajikistan. PLoS neglected tropical diseases. 2017;11(3):e0005367. doi: 10.1371/journal.pntd.0005367.
17.    Hamdy ME, Amin A. Detection of Brucella species in the milk of infected cattle, sheep, goats and camels by PCR. The Veterinary Journal. 2002;163(3):299-305. doi: 10.1053/tvjl.2001.0681. 
18.    Bonfini B, Chiarenza G, Paci V, Sacchini F, Salini R, Vesco G, et al. Cross-reactivity in serological tests for brucellosis: a comparison of immune response of Escherichia coli O157: H7 and Yersinia enterocolitica O: 9 vs Brucella spp. Vet Ital. 2018;54(2):107-14. doi: 10.12834/vetit.1176.6539.2.
19.    Ducrotoy MJ, Muñoz PM, Conde-Álvarez R, Blasco JM, Moriyón I. A systematic review of current immunological tests for the diagnosis of cattle brucellosis. Preventive veterinary medicine. 2018;151:57-72. doi: 10.1016/j.prevetmed.2018.01.005.
20.    Muñoz P, Marín C, Monreal D, Gonzalez D, Garin-Bastuji B, Diaz R, et al. Efficacy of several serological tests and antigens for diagnosis of bovine brucellosis in the presence of false-positive serological results due to Yersinia enterocolitica O: 9. Clinical and Vaccine Immunology. 2005;12(1):141-51. doi: 10.1128/CDLI.12.1.141-151.2005.
21.    Garin-Bastuji B, Blasco J. Caprine and ovine brucellosis (excluding B. ovis). Manual of diagnostic tests and vaccines for terrestrial animals: OIE. 2004:598-606.
22.    Smits HL, Abdoel TH, Solera J, Clavijo E, Diaz R. Immunochromatographic Brucella-specific immunoglobulin M and G lateral flow assays for rapid serodiagnosis of human brucellosis. Clinical and Vaccine Immunology. 2003;10(6):1141-6. doi: 10.1128/cdli.10.6.1141-1146.2003.
23.    Wormser GP, Pourbohloul B. Modeling Infectious Diseases in Humans and Animals By Matthew James Keeling and Pejman Rohani Princeton, NJ: Princeton University Press, 2008. 408 pp., Illustrated. $65.00 (hardcover). The University of Chicago Press; 2008. doi: 10.1515/9781400841035.
24.    Lalsiamthara J, Lee JH. Development and trial of vaccines against Brucella. Journal of Veterinary Science. 2017;18(S1):281-90. doi: 10.4142/jvs.2017.18.S1.281.  
25.    Refai M. Incidence and control of brucellosis in the Near East region. Veterinary microbiology. 2002;90(1-4):81-110. doi: 10.1016/s0378-1135(02)00248-1.
26.    Godfroid J, Cloeckaert A, Liautard J-P, Kohler S, Fretin D, Walravens K, et al. From the discovery of the Malta fever's agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis. Veterinary research. 2005;36(3):313-26. doi: 10.1051/vetres:2005003.
27.    Gwida M, Al Dahouk S, Melzer F, Rösler U, Neubauer H, Tomaso H. Brucellosis–regionally emerging zoonotic disease? Croatian medical journal. 2010;51(4):289-95. doi: 10.3325/cmj.2010.51.289.
28.    Blasco JM, Molina-Flores B. Control and eradication of Brucella melitensis infection in sheep and goats. Veterinary Clinics: Food Animal Practice. 2011;27(1):95-104. doi: 636.3.09:615.371.
29.    Banai M. Control of small ruminant brucellosis by use of Brucella melitensis Rev. 1 vaccine: laboratory aspects and field observations. Veterinary microbiology. 2002;90(1-4):497-519. doi: 10.1016/s0378-1135(02)00231-6. 
30.    Castelo C, Simões J. Risk factors of brucellosis (re-) incidence in sheep and goat flocks in an endemic area of Portugal. Tropical animal health and production. 2019;51:487-90. doi: 10.1007/s11250-018-1706-4.
31.    Pokorska J, Kułaj D, Dusza M, Żychlińska-Buczek J, Makulska J. New rapid method of DNA isolation from milk somatic cells. Animal biotechnology. 2016;27(2):113-7. doi: 10.1080/10495398.2015.1116446.
32.    Al-Talafhah AH, Lafi SQ, Al-Tarazi Y. Epidemiology of ovine brucellosis in Awassi sheep in Northern Jordan. Preventive Veterinary Medicine. 2003;60(4):297-306. doi: 10.1016/S0167-5877(03)00127-2.
33.    Samadi A, Ababneh MM, Giadinis ND, Lafi SQ. Ovine and Caprine Brucellosis (Brucella melitensis) in Aborted Animals in Jordanian Sheep and Goat Flocks. Vet Med Int. 2010 Oct 28;2010:458695. doi: 10.4061/2010/458695.
34.    Zhang H, Shengnan S, Benben W, Jiang Y, Wenxing W, Fei G, et al. Brucella melitensis isolated from aborted cow and sheep fetuses in northwest of China. Kafkas Üniversitesi Veteriner Fakültesi Dergisi. 2018;24(2). doi: 10.9775/kvfd.2017.18881
35.    Altun SK, Yiğin A, Gürbilek SE, Gürbüz S, Demirci M, Keskin O, et al. An enzyme-linked immunosorbent assay for Brucella specific antibody and real-time PCR for detecting Brucella spp. in milk and cheese in Şanlıurfa, Turkey. Pak Vet J. 2017;37(1):39-42. doi: 10.1099/00222615-13-1-167. 
36.    Sabrina R, Mossadak HT, Bakir M, Asma M, Khaoula B. Detection of Brucella spp. in milk from seronegative cows by real-time polymerase chain reaction in the region of Batna, Algeria. Veterinary world. 2018;11(3):363. doi: 10.14202/vetworld.2018.363-367.
37.    Zakaria AM. Comparative assessment of sensitivity and specificity of rose bengal test and modified in-house ELISA by using IS711 TaqMan Real Time PCR assay as a gold standard for the diagnosis of bovine brucellosis. Biomedical and Pharmacology Journal. 2018;11(2):951-7. doi: 10.13005/bpj/1453.
38.    Leal-Klevezas DS, Martínez-Vázquez IO, Lopez-Merino A, Martínez-Soriano JP. Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. Journal of clinical microbiology. 1995;33(12):3087-90. doi: 10.1128/jcm.33.12.3087-3090.1995.
39.    Romero C, Pardo M, Grillo MJ, Diaz R, Blasco J, Lopez-Goñi I. Evaluation of PCR and indirect enzyme-linked immunosorbent assay on milk samples for diagnosis of brucellosis in dairy cattle. Journal of clinical microbiology. 1995;33(12):3198-200. doi:10.1128/jcm.33.12.3198-3200.1995.
40.    Abdoel T, Dias IT, Cardoso R, Smits HL. Simple and rapid field tests for brucellosis in livestock. Veterinary Microbiology. 2008;130(3-4):312-9. doi: 10.1016/j.vetmic.2008.01.009.
41.    Sutherland S, Evans R, Bathgate J. Application of an enzyme‐linked immunosorbent assay in the final stages of a bovine brucellosis eradication program. Australian veterinary journal. 1986;63(12):412-5. doi: 10.1111/j.1751-0813.1986.tb15920.x .
42.    Lee CCK, Clarke I. Use of an enzyme‐linked immunosorbent assay in a bovine brucellosis eradication program. Australian veterinary journal. 1985;62(2):49-52. doi: 10.1111/j.1751-0813.1985.tb14233.x.
43.    Ferreira AC, Cardoso R, Dias IT, Mariano I, Belo A, Preto IR, et al. Evaluation of a modified Rose Bengal test and an indirect Enzyme-Linked Immunosorbent Assay for the diagnosis of Brucella melitensis infection in sheep. Veterinary Research. 2003;34(3):297-305. doi: 10.1051/vetres:2003005.
44.    De Miguel M, Marín CM, Muñoz PM, Dieste L, Grilló MJ, Blasco JM. Development of a selective culture medium for primary isolation of the main Brucella species. Journal of clinical microbiology. 2011;49(4):1458-63. doi:10.1128/jcm.02301-10.
45.    Queipo-Ortuño MI, De Dios Colmenero J, Macias M, Bravo MJ, Morata P. Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis. Clinical and Vaccine Immunology. 2008;15(2):293-6.  doi:: 10.1128/CVI.00270-07
46.    Kadivar A, Hassanpour H, Mirshokraei P, Azari M, Gholamhosseini K, Karami A. Detection and quantification of cell-free fetal DNA in ovine maternal plasma; use it to predict fetal sex. Theriogenology. 2013;79(6):995-1000. doi: 10.1016/j.theriogenology.2013.01.027.
47.    Richtzenhain LJ, Cortez A, Heinemann MB, Soares RM, Sakamoto SM, Vasconcellos SA, et al. A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses. Veterinary Microbiology. 2002;87(2):139-47. doi: 10.1016/S0378-1135(02)00049-4.
48.    García-Yoldi D, Marín CM, de Miguel MJ, Munoz PM, Vizmanos JL, López-Goñi I. Multiplex PCR assay for the identification and differentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1. Clinical chemistry. 2006;52(4):779-81. doi: 10.1373/clinchem.2005.062596.
49.    Landis JR, Koch GG. The measurement of observer agreement for categorical data. biometrics. 1977:159-74. doi: 10.2307/2529310.