Development of a novel multiplex PCR for detection of Brucella, Leptospira and Bovine herpesvirus-1

S K BHURE; S CHANDAN; R G AMACHAWADI; S S PATIL; RAJESWARI SHOME; N L GANGADHAR; M R GAJENDRA; K PRABHUDAS

doi:10.56093/ijans.v82i11.25127

Authors

S K BHURE Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India
S CHANDAN Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India
R G AMACHAWADI Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India
S S PATIL Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India
RAJESWARI SHOME Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India
N L GANGADHAR Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India
M R GAJENDRA Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India
K PRABHUDAS Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India

https://doi.org/10.56093/ijans.v82i11.25127

Keywords:

Bovine Herpesvirus-1, Brucella, Leptospira, Infectious bovine rhenotracheitis, Multiplex PCR

Abstract

A novel multiplex PCR (mPCR) was developed for detection of Brucella, Leptospira and bovine herpesvirus-1 (BoHV-1) targeting conserved regions of BCSP31, LipL32 and gB genes, respectively. No template controls were also run with each test. Electrophoresis of mPCR products showed bands specific to genus Brucella, Leptospira and BHV- 1, respectively. The assay detected as low as 15 pg of each template. The mPCR showed specific bands with 4 Brucella species, 14 pathogenic Leptospira serovars, BoHV-1. In addition, 10 (out of 135 bull semen samples) and 5 (out of 17 bovine blood samples) showed a ~640bp amplicon specific to brucellosis. Five human urine samples were tested; 3 found positive for Leptospira and other 2 were positive for both Leptospira and Brucella. The assay is accurate, specific and cost effective tool for detection of Brucella, Leptospira and bovine herpesvirus-1 and will also be a valuable tool for the diagnoses of co-infection. The assay could be a preferable detection methodology for routine screening and molecular epidemiology of brucellosis, leptospirosis and infectious bovine rhenotracheitis.

Downloads

Download data is not yet available.

Author Biographies

S K BHURE, Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India
S CHANDAN, Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India
R G AMACHAWADI, Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India
S S PATIL, Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India
RAJESWARI SHOME, Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India
N L GANGADHAR, Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India
M R GAJENDRA, Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India
K PRABHUDAS, Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, Karnataka 560 024, India

References

Amin A S, Hamdy M E and Ibrahim A K. 2001. Detection of Brucella melitensis in semen using the polymerase chain reaction assay. Veterinary Microbiology 83: 37–44,

Bhure S K, Lakshmisha K V, Patil S S, Rajeswari S, Shome B R, Gangadhar N L, Gajendragad M R and Pabhudas K. 2009. Rapid polymerase chain reaction (PCR) for detection of pathogenic Leptospires in biological samples. Indian Journal of Animal Sciences 79: 781–83.

Bomfim M R, Barbosa-Stancioli E F and Koury M C. 2008. Detection of pathogenic Leptospires in urine from naturally infected cattle by nested PCR. Veterinary Journal 178:251–56. Bounaadja L, Albert D, Chénais B, Hénault S, Zygmunt M S, Poliak S and Garin-Bastuji B. 2009. Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes. Veterinary Microbiology 137: 156–64.

Cândido A, Bontempo E and Resende M. 2000. Polymerase chain reaction amplification of genomic fragments of bovine herpesvirus-1. Memorias do InstitutoOswaldo Cruz 95: 225– 26.

Clause M P, Alfieri A F, Folgueras-Flatschart A V, Wosiacki SR, Medici K C and Alfieri A. 2005. A rapid detection and differentiation of bovine herepesvirus 1 and 5 glycoprotein C gene in clinical specimens by multiplex-PCR. Journal of Virological Methods 128: 183–88.

Cutler S J, Whatmore A M and Commander N J. 2005. Brucellosis – new aspects of an old disease. Journal of Applied Microbiology 98:1270–81.

Deka D, Ramneek M N K and Oberoi M S. 2005. Detection of bovine herpesvirus-1 infection in breeding bull semen by virus isolation and polymerase chain reaction. Revue Scientifeque et Technique (International office of Epizootics) 24: 1085–94.

Dutta T K and Christopher M. 2005. Leptospirosis – An Overview. The Journal of the association of Physicians of India 53:545– 51.

Eaglesome M D and Garcia M M. 1992. Microbial agents associated with bovine genital tract infections and semen. Part 1. Brucella abortus, leptospira, Campylobacter fetus and Tritrichomonas foetus. Veterinary Bulletin 62: 743–75.

Ellis W A, Cassels J A and Doyle J. 1986. Genital leptospirosis in bulls. Veterinary Record 118, 333.

Gangadhar N L, Prabhudas K, Bhushan S, Sulthana M, Barbuddhe S B and Rehaman H. 2008. Leptospira infection in animals and humans: a potential public health risk in India. Revue Scientifeque et Technique (International office of Epizootics) 27: 885–92.

Gibbs E P J and Rweyemamu M M. 1977. Bovine herpesviruses. Part I. Bovine herpesvirus 1. Veterinary Bulletin 47: 317–43.

Grom J, Hostnik P, Toplak I and Barlic-Maganja D. 2006. Molecular detection of BHV-1 in artificially inoculated semen and in the semen of a latently infected bull treated with dexamethasone. Veterinary Journal 171: 539–44.

Haake D A, Chao G, Zuerner R L, Barnett J K, Barnett D, Mazel M, Matsunaga J, Levett P N and Bolin C A. 2000. The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection. Infection and Immununity 68: 2276–85.

Karsen H, Karahocagil M K, Irmak H and Demiröz A P. 2008. A meningitis case of Brucella and tuberculosis co-infection. Mikrobiyoloji Bulteni 42: 689–94.

Kataria R S, Tiwari A K, Gupta P K, Mehrotra M L, Rai A and Bandyopadhyay S K. 1997. Detection of bovine herpesvirus 1 (BHV-1) genomic sequences in bovine semen inoculated with BHV-1 by polymerase chain reaction. Acta Virologica 41: 311- 15.

Kee S H, Kim I K, Choi M S and Chang W H. 1994. Detection of leptospiral DNA by PCR. Journal of Clinical Microbiology 32: 1035–39.

Levett P N. 2001. Leptospirosis. Clinical Microbiology Reveiw 14:296–26.

Merien F, Amouriauz P, Perolat P, Baranton G and Saint Girons I. 1992. Polymerase chain reaction for detection of Leptospira species in clinical samples. Journal of Clinical Microbiology 30: 2219–24.

Morata P, Queipo-Ortuño M I, Reguera J M, García-Ordoñez M A, Cárdenas A, and Colmenero J D. 2003. Development and evaluation of a PCR–enzyme-linked immunosorbent assay for diagnosis of human brucellosis. Journal of Clinical Microbiology 41: 144–48.

Navarro E, Escribano J, Fernandez J and Solera J. 2002. Comparison of three different PCR methods for detection of Brucella spp. in human blood samples. FEMS Immunology and Medical Microbiology 34: 147–51.

Probert W S, Schrader K N, Khuong N Y, Bystrom S L and Graves M H. 2004. Real-Time multiplex PCR assay for detection of Brucella spp, B. abortus and B. melitensis. Journal of Clinical Microbiology 42: 1290–93.

Rijpens N P, Jannes G, Van Asbroeck M, Rossau R and Herman L M. 1996. Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S–23S rRNA spacer probe. Applied and Environmental Microbiology 62: 1683–88.

Rola J, Miroslaw P and Polak Jan F Zmudzinski. 2003. Amplification of DNA of BHV1 isolated from semen of naturally infected bulls. Bulletin of Veterinary Institute in Pulawy 47:71–75.

Romero C, Gamazo C, Pardo M, López-Goñi I. 1995. Specific detection of Brucella DNA by PCR. Journal of Clinical Microbiology 33:615–17.

Silva E F, Brod C S, Cerqueira G M, Bourscheidt D, Seyffert N, Queiroz A, Santos C S, Ko A I and Dellagostin O A. 2007. Isolation of Leptospira noguchii from sheep. Veterinary Microbiology 121: 144–49.

Sohn A H, Probert W S, Glaser C A, Gupta N, Bollen A W, Wong J D, Grace E M and McDonald W C. 2003. Human neurobrucellosis with intracerebral granuloma caused by a marine mammal Brucella spp. Emerging Infectious Diseases 9: 485–88.

van Eys G J J M, Gravekamp C, Gerritsen M J, Quint W, Cornelissen M T E, Schegget J and Terpstra W J. 1989. Detection of Leptospires in urine by polymerase chain reaction. Journal of Clinical Microbiology 2:2258–62.

Zuerner R L, Alt D and Bolin C A. 1995. IS1533–based PCR assay for identification of Leptospira interrogans sensu lato serovars. Journal of Clinical Microbiology 33: 3284–89.