Molecular typing of Brucella species in dairy farms in the Province of Azuay – Ecuador

• Omar Santiago Andrade-Guzmán Universidad de Cuenca, Facultad de Ciencias Agropecuarias, Laboratorio de Microbiología y Lácteos. Cuenca, Ecuador
• Antonio Javier Vallecillo Universidad de Cuenca, Facultad de Ciencias Agropecuarias, Laboratorio de Biología Molecular. Cuenca, Ecuador
• Andrea Elizabeth Vintimilla-Rojas Universidad de Cuenca, Facultad de Ciencias Agropecuarias, Laboratorio de Microbiología y Lácteos. Cuenca, Ecuador
• Andrés Norberto Haro-Haro Universidad de Cuenca, Facultad de Ciencias Agropecuarias, Laboratorio de Microbiología y Lácteos. Cuenca, Ecuador
• Ivanna Solmayra Agreda-Orellana Universidad de Cuenca, Facultad de Ciencias Médicas, Laboratorio de Microbiología. Cuenca, Ecuador
• Daniela Alejandra Vintimilla-Rojas Universidad de Cuenca, Facultad de Ciencias Médicas. Cuenca, Ecuador
• Sergio Emiro Rivera-Pirela Universidad del Zulia, Facultad de Ciencias Veterinarias. Maracaibo, Zulia, Venezuela

DOI: https://doi.org/10.52973/rcfcv-e33239

Keywords: PCR-AMOS, B. abortus, IS711, ELISA–I, ELISA–C

Abstract

Brucellosis is a worldwide zoonotic disease in cattle, caused mainly by Brucella abortus previously reported in certain regions of Ecuador. The characterization of circulating strains of B. abortus in dairy herds is vital to understand the epidemiology of this disease. The bacteriological typing is a slow, risky process and requires specialized laboratories for Brucella spp. The aim of this study was to type the strains of Brucella spp. that affect bovines in the Province of Azuay, through molecular assays, from blood and milk samples from cows seropositive to brucellosis. In farms seropositive to ELISA-Indirect in milk, 70 Holstein crossbred cows were selected, individually reacting to the Rose Bengal tests and confirmed with competitive ELISA. The DNA was extracted from these blood and milk samples, initially verifying the viability of the bovine genetic material with specific oligonucleotides for the Bos genus. The DNA amplification for Brucella spp. It was performed by PCR-AMOS with genus primers for the IS711 region and Species primers for Brucella abortus, Brucella mellitensis, Brucella suis and Brucella ovis. Bos DNA could be identified in 65 (92.8%) milk samples and in 62 (88.5%) blood samples. A total of 62 (95.4%) DNA samples extracted from milk were positive for the genus Brucella spp. All blood samples were negative. The PCR-AMOS showed bands with a molecular weight of 498 base pairs in samples from four animals corresponding to B. abortus. This is the first study of molecular identification in the Province of Azuay with scientific evidence of the species of Brucella spp. circulating in the herds of the area, serving as a basis for the future identification of B. abortus biovars not yet reported in this area of the Country.

References

AL DAHOUK, S.; KÖHLER, S.; OCCHIALINI, A.; JIMÉNEZ DE BAGÜÉS, M. P.; HAMMERL, J. A.; EISENBERG, T.; VERGNAUD, G.; CLOECKAERT, A.; ZYGMUNT, M.S.; WHATMORE, A.M.; MELZER, F.; DREES, P.; FOSTER, T.; WATTAM, A.R.; SCHOLZ, H. C. Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts. Sci. Rep. 16(7) e44420. 2017. https://doi.org/f9t5z2

ABD AL-AZEEM, M.W.; MUSTAFA ELMALT, L.; ZAIN EL ABDEIN, A. E.; HELMY SAYED, H. Molecular and serological studies on detection of Brucella species in cattle and buffaloes. J. Pharm. Biomed. Sci. 2(3): 16–24. 2012. https://bit.ly/40J8Gxd. 20/02/2023

ALIYEV, J.; ALAKBAROVA, M.; GARAYUSIFOVA, A.; OMAROV, A.; ALIYEVA, S.; FRETIN, D.; GODFROID, J. Identification and molecular characterization of Brucella abortus and Brucella melitensis isolated from milk in cattle in Azerbaijan. BMC Vet. Res. 18(1): 1–9. 2022. https://doi.org/gp9q9b

ANDRADE, O.; LÓPEZ, M.; VINTIMILLA, A.; LUPERCIO, R.; BUSTAMANTE, J.; DUTÁN, J.; RIVERA, S.; GUEVARA, G. Eficacia de la prueba ELISA en muestras de leche para la vigilancia epidemiológica de la brucelosis bovina. Arch. Latinoame. Prod. Anim. 30(2): 67–69. 2022. https://doi.org/j7np

AWAH-NDUKUM, J.; MOUICHE, M.M.M.; KOUONMO-NGNOYUM, L.; BAYANG, H.N.; MANCHANG, T.K.; POUEME, R.S.N.; KOUAMO, J.; NGU-NGWA, V.; ASSANA, E.; FEUSSOM, K. J. M.; ZOLI, A.P. Seroprevalence and risk factors of brucellosis among slaughtered indigenous cattle, abattoir personnel and pregnant women in Ngaoundéré, Cameroon. B.M.C. Infect. Dis. 18(1): 1–13. 2018. https://doi.org/gj48rn

BRICKER, B.J.; HALLING, S.M. Differentiation of Brucella abortus bv. 1,2 and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. J. Clin. Microbiol. 32(11): 2660–2666. 1994. https://doi.org/j8dx

BRICKER, B.J.; HALLING, S.M. Enhancement of the Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51. J. Clin. Microbiol. 33(6): 1640–1642. 1995. https://doi.org/j8dz

CARBONERO, A.; GUZMÁN, L. T.; GARCÍA-BOCANEGRA, I.; BORGE, C.; ADASZEK, L.; ARENAS, A.; SAA, L.R. Seroprevalence and risk factors associated with Brucella seropositivity in dairy and mixed cattle herds from Ecuador. Trop. Anim. Health Product. 50(1): 197–203. 2018. https://doi.org/gcxdsz

DAL, T.; KARA, S.S.; CIKMAN, A.: BALKAN, C.E.; ACIKGOZ, Z.C.; ZEYBEK, H.; DURMAZ, R. Comparison of multiplex real-time polymerase chain reaction with serological tests and culture for diagnosing human brucellosis. J. Infect. Publ. Health. 12(3): 337–342. 2019. https://doi.org/gj48qt

DOGANAY, M.; AYGEN, B. Human brucellosis: An overview. Internat J. Infect. Dis. 7 (3): 173–182. 2003. https://doi.org/djsgvp

FERO, E.; JUMA, A.; KONI, A.; BOCI, J.; KIRANDJISKI, T.; CONNOR, R.; WARETH, G.; KOLECI, X. The seroprevalence of brucellosis and molecular characterization of Brucella species circulating in the beef cattle herds in Albania. PLoS ONE. 15(3): 1–14. 2020. https://doi.org/gj48ff.

GHAHERI, M.; KAHRIZI, D.; YARI, K.; BABAIE, A.; SUTHAR, R.S.; KAZEMI, E. A comparative evaluation of four DNA extraction protocols from whole blood sample. Cell. Mol. Biol. 62(3): 119–123. 2016. https://doi.org/j8d3.

GODFROID, J.; SCHOLZ, H.C.; BARBIER, T.; NICOLAS, C.; WATTIAU, P.; FRETIN, D.; WHATMORE, A.M.; CLOECKAERT, A.; BLASCO, J.M.; MORIYON, I.; SAEGERMAN, C.; MUMA, J.B.; AL DAHOUK, S.; NEUBAUER, H.; LETESSON, J.J. Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century. Prev. Vet. 102(2): 118–131. 2011. https://doi.org/c39d67

GOUD, T.S.; UPADHYAY, R.C.; KUMAR, A.; KARRI, S.; CHOUDHARY, R.; ASHRAF, S.; SINGH, S.; KUMAR, O.; KIRANMAI, C. Novel extraction of high quality genomic dna from frozen bovine blood samples by using detergent method. Open Vet. J. 8(4): 415–422. 2018. https://doi.org/gfrxmg

HALLING, S.M.; TATUM, F.M.; BRICKER, B.J. Sequence and characterization of an insertion sequence, IS711, from Brucella ovis. Gene. 133(1): 123–127. 1993. https://doi.org/c3m6fx.

HAMDY, M.E.R.; AMIN, A.S. Detection of Brucella species in the milk of infected cattle, sheep, goats and camels by PCR. Vet. J. 163(3): 299–305. 2002. https://doi.org/fbhphh

KILIÇ, S.; ÇELEBI, B.; TURAN, M. Brucella mellitensis and Brucella abortus genotyping via real-time PCR targeting 21 variable genome loci. J. Microbiol. Meth. 180: 106125. 2021. https://doi.org/j8vb.

KURMANOV, B.; ZINCKE, D.; SU, W.; HADFIELD, T. L.; AIKIMBAYEV, A.; KARIBAYEV, T.; BERDIKULOV, M.; ORYNBAYEV, M.; NIKOLICH, M.P.; BLACKBURN, J.K. Assays for Identification and Differentiation of Brucella Species: A Review. Microorganisms. 10(8): 1584. 2022.. https://doi.org/j8vc.

LEAL-KLEVEZAS, D.S.; MARTÍNEZ-VÁZQUEZ, I.O.; LÓPEZ-MERINO, A.; MARTÍNEZ-SORIANO, J.P. Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. J. Clin. Microbiol. 33(12): 3087–3090. 1995. https://doi.org/j8vd.

LEDWABAID, M.B.; GOMO, C.; LEKOTA, K.E.; LE FLÈCHE, P.; HASSIM, A.; VERGNAUD, G.; VAN HEERDEN, H. Molecular characterization of Brucella species from Zimbabwe. PLoS Neglected Trop. Dis. 13(5): e0007311. 2019. https://doi.org/gqrrp7.

LÓPEZ-CALLEJA, I.; GONZÁLEZ, I.; FAJARDO, V.; RODRÍGUEZ, M.A.; HERNÁNDEZ, P.E.; GARCÍA, T.; MARTÍN, R. Rapid detection of cows' milk in sheeps' and goats' milk by a species-specific polymerase chain reaction technique. J. Dairy Sci. 87(9): 2839-2845. 2004. https://doi.org/fnqhxr.

LUNA. L.; CHÁVEZ, G.; MEJÍA, L.; BARRAGÁN, V.; TRUEBA, G. Molecular detection of Brucella species in Ecuador. Internat. J. Appl. Res. Vet. Med. 14(2): 185–189. 2016. https://bit.ly/3LGAtc1. 20/12/2022.

MAINATO, E.A.; RAMÓNEZ, J.C.; RAMÓNEZ, M.A.; GARAY, G.S. Factores de riesgo que influyen sobre la frecuencia de brucelosis bovina en ganaderías de Azuay, Ecuador. Arch. Latinoame. Prod. Anim. 30: 57–58. 2022. https://doi.org/j8vf.

MANCILLA, M.; VILLARROEL, M.; SALDÍAS, M.E.; SOTO, J.; ZÁRRAGA, A.M. Genotipos de aislados de campo de Brucella abortus de distintas regiones geográficas de Chile. Arch. Med. Vet. 40(2): 187–192. 2008. https://bit.ly/3M1W6oz. 20/12/2022.

MOSQUERA, X.; BERNAL, C.; MUSKUS, C.; BERDUGO, J. Detección de Brucella abortus por PCR en muestras de sangre y leche de vacunos. Rev. MVZ. Cordova. 13(3): 1504–1513. 2008. https://bit.ly/3nGWvmT.

OGUGA, A.J.; AKINSEYE, V.O.; CADMUS, E.O.; JOLAOLUWA-AWOSANYA, E.A.; ALABI, P.I.; IDOWU, O.S.; AKINADE, S.A.; DALE, E.J.; PERRETT, L.; TAYLOR, A.; IGNOCIO, M.; CADMUS, S.I.B. Prevalence and risk factors associated with bovine brucelosis in herds under extensive production system in southwestern Nigeria. Trop. Anim. Health Prod. 50(7): 1573–1582. 2018. https://doi.org/gfdk62.

OJEDA-GUTIERREZ, K.; ROMÁN-CÁRDENAS F. Identificación molecular de Brucella spp. en muestras de sangre de ganado bovino de la provincia de Zamora Chinchipe‏. Centro Biotecnol. 07: 11–16. 2018. https://bit.ly/44MJ6L0. 15/01/2023.

O'LEARY, S.; SHEAHAN, M.; SWEENEY, T. Brucella abortus detection by PCR assay in blood, milk and lymph tissue of serologically positive cows. Res. Vet. Sci. 81(2): 170–176. 2006. https://doi.org/dj6s4p.

OUAHRANI-BETTACHE, S.; JIMÉNEZ DE B, M.P.; DE LA GARZA, J.; FREDDI, L.; BUESO, J.P.; LYONNAIS, S.; AL DAHOUK, S.; DE BIASE, D.; KÖHLER, S.; OCCHIALINI, A. Lethality of Brucella microti in a murine model of infection depends on the wbkE gene involved in O-polysaccharide synthesis. Virulence. 10(1): 868–878. 2019. https://doi.org/grkg3z.

POULSEN, K.P.; HUTCHINS, F.T.; MCNULTY, C.M.; TREMBLAY, M.; ZABALA, C.; BARRAGAN, V.; LOPEZ, L.; TRUEBA, G.; BETHEL, J.W. Brucellosis in dairy cattle and goats in northern Ecuador. The Amer. J. Trop. Med. Hyg. 90(4): 712–715. 2014. https://doi.org/f5xmvd.

RAGHAVA, S.; GOWDA, M.H.M.; SHOME, R.; KULKARNI, M.; UMESHA, S. Epidemiological and molecular characterization of Brucella species in cattle. Asian. J. Anim. Sci. 11(3): 123–131. 2017. https://doi.org/j8vg.

RENTERÍA-EVANGELISTA, T.B.; ORGANES-DE LOS SANTOS, H.; LICEA-NAVARRO, A.F.; MEDINA-BASULTO, G.E.; NIELSEN, K.; MONTAÑO-GÓMEZ, M. F.; MORENO-ROSALES, J.F.; PUJOL-MANRÍQUEZ, L.C. Evaluación de la prueba reacción en cadena de la polimerasa (PCR) a partir de muestras de leche y cultivos puros en el diagnóstico de la brucelosis bovina. Téc. Pec. México. 43(1): 117–126. 2005. https://bit.ly/3NLbRkT. 03/01/2023.

RODRIGUEZ-HIDALGO, R I.; CONTRERAS-ZAMORA, J.; BENITEZ-ORTIZ, W.; GUERRERO-VIRACOCHA, K.; SALCAN-GUAMAN, H.; MINDA, E.; RON-GARRIDO, L. Circulating strains of Brucella abortus in cattle in Santo Domingo de los Tsachilas Province-Ecuador. Front. Publ. Health. 3(45): 1–5. 2015. https://doi.org/j8vj.

ROMÁN-CÁRDENAS, F.; LUNA-HERRERA, J. Revisión actualizada de la epidemiología de Brucelosis (Brucella abortus, Brucella mellitensis, Brucella suis, Brucella canis) en el Ecuador y el mundo. Centro Biotecnol. 6: 82–93. 2017. https://bit.ly/3B7IstD.

ROMAN-CARDENAS, F.; RAMÓN-CONTENTO, P. Identificación molecular de Brucella abortus en nódulos linfáticos de bovinos faenados en Loja. Siembra. 8 (1): 1–10. 2021. https://doi.org/j8vp.

ROMERO, C.; LOPEZ-GOÑI, I. Improved method for purification of bacterial DNA from bovine milk for detection of Brucella spp. by PCR. Appl. Environm. Microbiol. 65(8): 3735–3737. 1999. https://doi.org/j8vq.

RON-ROMÁN, J.; BERKVENS, D.; BARZALLO-RIVADENEIRA, D.; ANGULO-CRUZ, A.; GONZÁLEZ-ANDRADE, P.; MINDA-ALUISA, E.; BENÍTEZ-ORTÍZ, W.; BRANDT, J.; RODRÍGUEZ-HIDALGO, R.; SAEGERMAN, C. The unexpected discovery of Brucella abortus Buck 19 vaccine in goats from Ecuador underlines the importance of biosecurity measures. Trop. Anim. Health Prod. 49(3): 569–574. 2017. https://doi.org/f9x4hr.

RON-ROMÁN, J.; RON-GARRIDO, L.; ABATIH, E.; CELI-ERAZO, M.; VIZCAÍNO-ORDÓÑEZ, L.; CALVA-PACHECO, J.; GONZÁLEZ-ANDRADE, P.; BERKVENS, D.; BENÍTEZ-ORTÍZ, W.; BRANDT, J.; FRETIN, D.; SAEGERMAN, C. Human brucellosis in northwest Ecuador: typifying Brucella spp., seroprevalence and associated risk factors. Vector Borne Zoon. Dis. 14(2): 124–133. 2014. https://doi.org/j8vs.

SCHURIG, G.G.; ROOP, R.M.; BAGCHI, T.; BOYLE, S.; BUHRMAN, D.; SRIRANGANATHAN, N. Biological properties of RB51; a stable rough strain of Brucella abortus. Vet. Microbiol. 28(2): 171–188. 1991. https://doi.org/cpjcm8.

SCHWENKER, J.A.; FRIEDRICHSEN, M.; WASCHINA, S.; BANG, C.; FRANKE, A.; MAYER., R.; HÖLZEL, S. Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples. Microbiol. Open. 11(2): e1275. 2022. https://doi.org/j8vt.

SUÁREZ-ESQUIVEL, M.; RUIZ-VILLALOBOS, N.; JIMÉNEZ-ROJAS, C.; BARQUERO-CALVO, E.; CHACON-DIAZ, C.; VIQUE-RUIZ, E.; ROJAS-CAMPOS, N.; BAKER, S. K.; OVIEDO-SANCHEZ, G.; AMUY, E.; CHAVES-OLARTE, E.; THOMSON, N. R.; MORENO, E.; GUZMAN-VERRI, C. Brucella neotomae Infection in Humans, Costa Rica. Emerging Infectious Diseases. 23(6) 997–1000. 2017. https://doi.org/gbhb6k.

TORRES-HIGUERA, L.D.; JIMÉNEZ-VELÁSQUEZ, S.D.C.; RODRÍGUEZ-BAUTISTA, J.L.; PATIÑO-BURBANO, R.E. Identification of Brucella abortus biovar 4 of bovine origin in Colombia. Rev. Argentina Microbiol. 51(3): 221–228. 2019. https://doi.org/j8vw.

WURYASTUTY, H.; WASITO, R.; SUGIYONO. Molecular identification of Brucella abortus collected from whole blood samples of seronegative dairy cattle with reproductive disorders in Central Java, Indonesia. Pakistan Vet. J. 39(3): 455–458. 2019. https://doi.org/j8vx.