Isolation and Molecular Characterization of BCSP-31 Gene of Brucella abortus from Bovine Abortions.
Brucellosis is an infectious disease with considerable impact on animals as well as public health. It causes significant reproductive losses in cattle directly affecting production. Brucellosis is endemic in many developing countries and is caused by Brucella species that affect man, domestic animals, some wild animals and marine mammals (Bhatia and Narain, 2010; Seleem et al., 2010 and Geresu et al., 2016). An estimated 5,00,000 new human Brucella cases are reported annually worldwide (Pappas et al., 2006). Brucellosis is the second most important zoonosis after rabies. Brucella species are gram negative cocco-bacilli, which are classified into species by various techniques such as growth patterns on media and phage susceptibility.
There are six 'classical' recognized species, B. abortus, B. melitensis, B. suis, B. ovis, B. canis and B. neotomae (Godfroid et al., 2005; Hadush and Pal, 2013). Sources of infection for transmission of bovine brucellosis are aborted fetuses, fetal membranes after birth, vaginal discharges and milk from infected animals (Tolosa et al., 2010 and Geresu et al., 2016). BCSP31 gene characterized in our study is protein that frequently elicits an immune response during infection (Pugh et al., 1989) and has been candidate in making DNA vaccines (Yu et al., 2007). Materials and Methods
Samples (on ice) received from two different cases of abortion from an organized dairy farm were taken for bacterial isolation, identification and further molecular characterization by PCR. Pieces of aborted placenta and cervical swab of dam were received from one case of abortion whereas fetal heart blood and stomach content of aborted fetus were received from other case of abortion. Serum samples (on ice) of both dams were also received for serological examination. All four clinical samples received on ice were plated on Blood agar (a) (containing 5% Sheep RBCs), Brucella selective agar (a) [containing 5% v/v inactivated horse serum (RM1239) and Brucella selective supplement (FD005)] and on MaConkey agar (a) and incubated anaerobically in an atmosphere of 5-10% C[O.sub.2] for seven days. The plates were observed at every 24 hours interval for growth for bacteriological isolation. The serum samples were also screened for positive titer against Brucella abortus antigen (IVRI, Izatnagar) by Serum Agglutination test (SAT) and subsequently by Rose Bengal Plate test (RBPT) antigen (IVRI, Izatnagar). The isolated bacterial colonies were further confirmed by Gram stain and modified Ziehl-Nelsen stain (MZN) followed by biochemical tests and molecular characterization of BCSP-31 gene by PCR.
All four samples showed non-hemolytic, small, glistening, opaque colonies on blood agar plates and on Brucella selective agar bacterial colonies were pin-point, smooth, glistening and translucent after seven days of inoculation in an atmosphere of 5-10% C[O.sub.2] (Fig.1). In Gram's staining pink, gram negative, coccobacillary rods were observed (Fig. 2). While in MZN staining they appeared to be red coccobacillary (Fig. 3). Absence of growth on Mac Conkey agar was also observed. All these four (4) isolates gave positive reaction in catalase (Fig. 4) and oxidase tests (Fig. 5). On TSI slant, organism showed reaction as slant (yellow), butt (black) (Fig. 6) and on urea slant pink colour (Fig. 7) indicative as Brucella abortus.
The serum samples from both dams showed positive titre of 1:80 (160 IU) in serum agglutination test and rapid agglutination when tested against Rose Bengal Plate Test (RBPT) antigen (Fig.8).
Molecular Detection of Brucella abortus DNA Extraction: DNA extraction was carried out from clinical samples and colony using DNeasy Blood and Tissue Kit (Qiagen) following manufacturers protocols.
PCR Amplification: The primer Forward 5' GGGCAA GGT GGA AGA TTT 3' and Reverse 5'CGG CAA GGG TCG GTG TTT 3' used in this study were from the OMP BCSP-31 gene sequence (Mayfield et al., 1988). The conditions for amplification were; initial denaturation at 94[degrees]C for three min followed by 35 cycles of 94[degrees]C for 45 sec and 53[degrees]C for 1 min, DNA extension at 72[degrees]C for 1 min with final extension at 72[degrees]C for 5 min. Finally, the PCR amplicon was confirmed on 1.5% (w/v) agarose gel stained with ethidium bromide and visualised on UV transilluminator /Geldoc system. A positive result was indicated by separation of amplicon of 443 bp (Fig. 9). Bacterial colonies of all four clinical samples yielded product size of 443 bp on PCR amplification.
Nucleotide sequence accession number
The sequence of Brucella abortus BCSP-31 isolated from cervical swab reported here has been submitted in International Gene Bank, NCBI, Bethseda (USA) and assigned Gen Bank accession number KY287764. Phylogenetic tree analysis of Brucella abortus BCSP-31 among related strains is shown in Fig. 10.
The diagnosis of brucellosis is based on serological, bacteriological and molecular methods (Simsek, 2004). The most important confirmatory diagnostic method of Brucella infection is bacteriological isolation since its specificity is much higher than that of other diagnostic methods and it is used as a gold standard diagnostic method. Because of the complications involved in diagnosis of disease, including difficulties in distinguishing between infected and vaccinated animals by conventional serological tests, bacteriological isolation and identification of biotypes of etiological agent are necessary steps in design of epidemiological and eradication programmes (Refai, 2002; Zinstag et al., 2005). Molecular diagnostic methods are also being used for detection of Brucella spp. in various samples (Sahin et al., 2008). Amplification of specific region of bacterial DNA using PCR has tremendous potential in diagnosis of Brucella.
In present study, bacteriological culture, morphological and biochemical tests confirmed that all four isolates obtained from placenta, stomach content, heart blood and cervical swabs were B. abortus. Koneman et al. (1997); Poester et al. (2006) and Lage et al. (2008) reported similar findings.
In consonance with earlier reports, all Brucella isolates found in our study were positive for catalase, oxidase and urea hydrolysis and negative for indole production revealing them to be Brucella (Koneman et al., 1997). The aborted fetus has been considered as one of the best sample to isolate Brucella in cattle and buffalo (Nielsen and Duncan, 1990). The serum samples of both dams showed positive titer in Serum Agglutunatiion test (SAT) Antigen and presence of agglutination in Rose Bengal Plate test (RBPT). Thus isolation of B. abortus from aborted fetuses and sero positive cattle with history of abortion is in line with findings by Gulhan et al. (2011) and Ali et al. (2014).
Molecular characterization of Brucella abortus isolates carried out in our study showed the presence of BCSP-31 gene which is a protein that frequently elicits an immune response during infection as reported by Pugh et al.,1989 and is a significant tool in manufacturing marker vaccine that make it possible to distinguish between vaccinated and infected animals. Further BCSP-31 gene characterized in our study has been candidate in making DNA vaccines (Yu et al., 2007). A combined DNA vaccine encoding BCSP31, SOD and L7/L12 confers high protection against Brucella abortus 2308 by inducing specific CTL responses as compared to other monovalent DNA vaccines, possible due to presence of brucella specific BCSP31, 31k Da immunodominant protein (Yu et al., 2007).
Bovine brucellosis caused by B. abortus has a major impact on human health, besides causing significant economic losses in dairy industry. A number of research reports on sero-prevalence, bacteriological isolation and molecular characterization of Brucella abortus has shown beyond doubt that disease is still prevalent in dairy cattle in India. The organisms were isolated from placenta, fetal stomach contents, fetal heart blood and cervical swab of dams. Hence, it is of practical importance to isolate Brucella spp. The use of polymerase chain reaction (PCR) to identify Brucella DNA at genus and species levels has becoming expedient to improve diagnostic tests. Further research are required to delineate the contribution of different immune pathways in response to B. abortus infection and development of a modified live brucellosis vaccine with distinctive immunological signature.
The authors are thankful to the DGRVS, QMG branch, IHQ of MOD for providing the necessary facilities to carry out the work.
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Chhabil Singh (1), Rahul Sharma (2), S.B. RamaRaju Sagi (2), Vikas Thakur (3) and Amit Kumar (4)
Central Military Veterinary Laboratory (CMVL) Sardhana Road Meerut Cantt.
Meerut - 250001 (Uttar Pradesh)
(1.) Lab Officer and Corresponding author. E-mail: firstname.lastname@example.org
(2.) Lab Officer
(4.) Dir RVS (Tech), RV Branch, IHQ of MoD
a - Brand of HiMedia Laboratories Ltd., Mumbai
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|Title Annotation:||Research Article|
|Author:||Singh, Chhabil; Sharma, Rahul; Sagi, S.B. Ramaraju; Thakur, Vikas; Kumar, Amit|
|Date:||Jul 1, 2018|
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