Abstract

The present investigation was undertaken to detect brucellosis in cattle using serological, bacteriological and molecular techniques. A total of 50 samples including milk (24), placenta (2), blood (22) and vaginal discharge (2) from cattle with a history of abortion were collected from farms located in and around Vasai region. The specimens were processed for detection of brucellosis by different methods: RBPT, MRT, bacteriology (cultural isolation and identification) and molecular techniques (BCSP 31 PCR, IS711/AB PCR assays). Out of 24 milk samples tested by MRT, 2 samples were found positive for the presence of Brucella antibodies with an overall prevalence of 8.33%. Whereas out of 22 sera samples of animals examined by RBPT, 5 (22.72%) were positive for the presence of Brucella antibodies. Three Brucella spp. isolates were recovered from 28 samples with a 10.71% isolation rate. Antibiotic sensitivity testing showed that all B. abortus isolates (100%) are sensitive to cefotaxime, doxycycline and gentamicin, and 66.66% of B. abortus isolates were resistant to erythromycin and streptomycin. The BCSP31 and IS711/AB PCR assay were carried out to confirm the identity of isolates recovered from clinical cases. The BCSP31 PCR showed an amplicon of 223 bp in all 03 isolates and reference strain B. abortus 544. The IS711/AB PCR analysis of the reference strain and all the isolates revealed a species amplicon of 498 bp. 

Reference

Abro SH, Khan M, Abro R, Leghari RA, Rizwana H et al., 2017. Antibiotic susceptibility of Brucella abortus isolated from milk and blood samples of cattle. J Basic Appl Sci, 13: 8-11, doi: 10.6000/1927-5129.2017.13.02

Ali S, Ali Q, Melzer F, Khan I, Akhter S et al, 2014. Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR. Trop Anim Health Prod, 46(1): 73-78, doi: 10.1007/s11250-013-0448-6

Al-Mashhadany DA, 2019. The significance of milk ring test for identifying Brucella antibodies in cows and buffaloes' raw milk at Erbil governorate, Iraq. Iraqi J Vet Sci, 33(2): 395-400, doi: 10.33899/IJVS.2019.163085

Alton GG, Jones LM, Angus RD and Verger JM, 1988. Bacteriological Methods. In Techniques for the Brucellosis Laboratory. INRA, Paris, pp 13-62

Baily GG, Krahn JB, Drasar BS and Stoker NG, 1992. Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg, 95(4): 271-275

Bounaadja L, Albert D, Chenais B, Henault S, Zygmunt MS et al., 2009. Real-time PCR for identification of Brucella spp.: A comparative study of IS711, BCSP31 and per target genes. Vet Microbiol, 137(1-2): 156-164, doi: 10.1016/j.vetmic.2008.12.023

Bricker BJ and Halling SM, 1994. 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, doi: 10.1128/jcm.32.11.2660-2666.1994

Chand P and Chhabra, R, 2013. Herd and individual animal prevalence of bovine brucellosis with associated risk factors on dairy farms in Haryana and Punjab in India. Trop Anim Health Prod, 45(6): 1313-1319, doi: 10.1007/s11250-013-0362-y

CLSI (Clinical and Laboratory Standards Institute), 2018. Performance standards for antimicrobial susceptibility testing. M100 28th edn. Wayne, PA

Da Costa M, Guillou JP, Garin?Bastuji B, Thiebaud M and Dubray G, 1996. Specificity of six gene sequences for the detection of the genus Brucella by DNA amplification. J Appl Bacterial, 81(3): 267-275, doi: 10.1111/j.1365-2672.1996.tb04328.x

Dadar M, Alamian S, Brangsch H, Elbadawy M, Elkharsawi AR et al., 2023. Determination of virulence-associated genes and antimicrobial resistance profiles in Brucella isolates recovered from humans and animals in Iran using NGS technology. Pathogens, 12(1): 82, doi: 10.3390/pathogens12010082

Geresu MA, Ameni G, Wubete A, Arenas-Gamboa AM and Kassa GM, 2016. Isolation and Identification of Brucella species from dairy cattle by biochemical tests: The first report from ethiopia. Worlds Vet J, 6(2): 80-88, doi: 10.5455/wvj.20160471

Jain A, Sharma NS, Kaur P and Arora AK, 2013. Isolation, biotyping and antibiogram evaluation of Brucella abortus in cattle and buffaloes from Punjab (India). Appl Biol Res, 15(1): 27-31

Kaur P, Sharma NS, Arora AK and Chachra D, 2018. Investigation of brucellosis in cattle and buffaloes by conventional and molecular assays. Indian J Anim Res, 52(10): 1482-1487, doi: 10.18805/ijar.B-3375

Khan I, Ali S, Hussain R, Raza A, Younus M et al., 2021 Serosurvey and potential risk factors of brucellosis in dairy cattle in peri-urban production system in Punjab, Pakistan. Pak Vet J, 10: 459-462

Khan MZ, Usman T, Sadique U, Qureshi MS, Farooque M et al., 2017. Molecular characterization of Brucella abortus and Brucella melitensis in cattle and humans at the north west of Pakistan. Pak Vet J, 37: 360-363

Lage AP, Poester FP, Paixao TA, Silva TA, Xavier MN et al., 2008. Brucelose bovina: uma atualizaçao. Rev Bras Reprod Anim Belo Horizonte, 32(3): 202-212

Leal-Klevezas DS, Martinez-Vazquez IO, Lopez-Merino A and Martinez-Soriano JP, 1995. Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. J Clin Microbiol, 33(12): 3087-3090, doi: 10.1128/jcm.33.12.3087-3090.1995

Londhe SP, Bannalikar AS and Dighe VD, 2010. Detection of Brucella abortus in buffalo blood and milk by IS711 PCR assay. J Vet Pub Hlth, 8(2): 95-98

Lucero N, Ayala SM, Escobar GI and Jacob NR, 2008. Brucella isolated in humans and animals in Latin America from 1968 to 2006. Epidemiol Infect, 136(4): 496-503, doi: 10.1017/S0950268807008795

Mugizi DR, Muradrasoli S, Boqvist S, Erume J, Nasinyama GW et al., 2015. Isolation and molecular characterization of Brucella isolates in cattle milk in Uganda. Biomed Res Int, 2015: 720413, doi: 10.1155/2015/720413

OIE, 2009. Office Internationale des Epizooties. Manual of Standards for Diagnostic Tests and Vaccines, 6th edn. France, OIE Press, pp 389-428

OIE, 2018. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. World Organization for Animal Health, pp 355-398

Poester FP, Gonçalves VSP, Paixao TA, Santos RL, Olsen SC et al., 2006. Efficacy of strain RB51 vaccine in heifers against experimental brucellosis. Vaccine, 24(25): 5327-5334, doi: 10.1016/j.vaccine.2006.04.020

Poester FP, Nielsen K, Samartino LE and Yu WL, 2010. Diagnosis of brucellosis. The Open Vet Sci J, 4: 46-60, doi: 10.2174/1874318801004010046

Probert WS, Schrader KN, Khuong NY, Bystrom SL and Graves MH, 2004. Real-time multiplex PCR assay for detection of Brucella spp., B. abortus, and B. melitensis. J Clin Microbiol, 42(3): 1290-1293, doi: 10.1128/JCM.42.3.1290-1293.2004

Quinn PJ, Carter ME, Markey B and Carter GR, 1994. Clinical Veterinary Microbiology. Wolfe publishing: London

Romero C, Gamazo C, Pardo M and Lopez-Goni I, 1995. Specific detection of Brucella DNA by PCR. J Clin Microbiol, 33(3): 615-617, doi: 10.1128/jcm.33.3.615-617.1995

Rossetti OL, Arese AI, Boschiroli ML and Cravero SL, 1996. Cloning of Brucella abortus gene and characterization of expressed 26-kilodalton periplasmic protein: potential use for diagnosis. J Clin Microbiol, 34(1): 165-169, doi: 10.1128/jcm.34.1.165-169.1996

Saleha S, Basit A, Rahim K, Shahid M and Khan MA, 2014. Comparison of milk ring test; serum plate agglutination test and polymerase chain reaction for the detection of bovine brucellosis. Res J Vet Pract, 2(1): 5-8, doi: 10.14737/journal.rjvp/2014/2.1.5.8

Schurig GG, Roop 2nd RM, Bagchi T, Boyle S, Buhrman D et al., 1991. Biological properties of RB51; a stable rough strain of Brucella abortus. Vet Microbiol, 28(2): 171-188, doi: 10.1016/0378-1135(91)90091-s

Selim A, Gaber A and Moustafa A, 2015. Diagnosis of brucellosis in ruminants in Kafr El-Sheikh governorate, Egypt. Int J Adv Res, 3(1): 345-350

Suryawanshi A and Gandge R, 2017. Detection and antibiotic sensitivity testing of Brucella species. World J Pharm Pharm Sci, 6(7): 1817-1829, doi: 10.20959/wjpps20177-9621

Tasiame W, Emikpe BO, Folitse RD, Fofie CO, Burimuah V et al., 2016. The prevalence of brucellosis in cattle and their handlers in North Tongu District of Volta Region, Ghana. Afr J Infect Dis, 10(2): 111-117, doi: 10.21010/ajid.v10i2.6