Res. Environ. Life Sci. 10(8) (2017)

2017 RELS ISSN: 0974-4908 http://rels.comxa.com Res. Environ. Life Sci. rel_sci@yahoo.com 10(8) 718-723 (2017) Isolation, biochemical characterization and PCR confirmation of antibiotic resistant Brucella abortus from cattle and buffaloes in Punjab state of India Vimlesh Gupta and Hari Mohan Saxena* Department of Veterinary Microbiology, College of Veterinary Science, GADVASU, Ludhiana 141004 India *e-mail: drhmsaxena@gmail.com (Received: October 09, 2016; Revised received: April 08, 2017; Accepted: April 14, 2017) Abstract: Brucellosis is a highly contagious and important zoonotic disease worldwide and is endemic in India. Antibiotics are currently used in treatment of Brucellosis. However, little information is available on the antibiotic sensitivity of field isolates of Brucella in different parts of India. We analyzed clinical samples (foetal stomach contents, foetal membranes, uterine fluid, vaginal mucous and milk) from cattle and buffaloes from Ludhiana for Brucella abortus organisms. Out of the total 104 samples analyzed, 12 were positive for Brucella abortus by isolation. The isolates were confirmed to be Brucella organisms by PCR. Morphological characterstics like smooth, glistening, translucent and pinpoint colonies appearing after 3-5 days of incubation in microaerophilic environment were indicative of Brucella. The organisms were found to be positive for catalase, oxidase and urease. H 2 S was produced by 7 isolates. All the 12 isolates were positive for nitrate reduction and were negative for indole test. All the 12 isolates of Brucella abortus were found to be positive by PCR using Brucella specific primers B4/B5 and F4/R2 which revealed the desired amplicons of 223 bp and 905 bp, respectively. Isolated organisms were sensitive to chloramphenicol, oxytetracyclin, ampicillin, streptomycin, gatifloxacin and ceftaxime but resistant to penicillin, cotrimazole, cephalothin and erythromycin. Keywords: Brucella abortus, isolation, antibiotic resistant, Brucellosis. Introduction Brucellosis is a highly contagious and important zoonotic disease caused by organisms of the genus Brucella. These are pathogenic for a wide variety of animals such as swine, cattle, goat, sheep, and dogs and also for humans (Mathur et al 2007). In animals, brucellosis mainly affects reproduction and fertility, reduces the sur vival of newborns, and diminishes milk yield. Outbreak of brucellosis in animals is characterized by abortions during the last trimester of gestation. While animals typically recover, and will be able to have live offspring following the initial abortion, they may continue to shed the bacteria (Sewel and Blocklesby 1990). Death may occur as a result of acute metritis followed by retained foetal membranes. Brucellosis is endemic in India and is prevalent in all parts of the country. In Punjab, prevalence of brucellosis has been found to be 17.68% (Aulakh et al 2008) and is considered the major cause of abortion in animals. High prevalence of brucellosis up to 23.2% has been recorded in the districts of Moga, Ludhiana, Amritsar and Kapurthala in Punjab. Ever increasing antibiotic resistance among various pathogenic bacteria in the country (Qureshi and Saxena, 2014) is alarming. We, therefore, tested field isolates of Brucella abortus to explore for any resistance to common antibiotics among them. Materials and Methods Source and collection of samples: Samples were collected from animals reporting to Teaching Veterinary Clinical Complex Research in Environment and Life Sciences 718 and Dairy Farm of GADVASU and Hara Dairy Farm, Ludhiana, respectively. In the present study a total of 104 samples from brucellosis suspected bovines (78 from cattle and 26 from buffaloes) were collected. Samples from cattle included foetal stomach contents (FSC 10), uterine fluid (24), milk (35), foetal membranes (2), and vaginal mucous (7). Samples from buffaloes comprised foetal stomach contents (4), uterine fluid (6), milk (14), and vaginal mucous (2) (Table 5). Samples were collected in sterile containers from different dairy farms in and around Ludhiana and were immediately transported on ice to the laboratory for further processing. Processing of samples and isolation of Brucella: For primary isolation of Brucella, the Brucella selective medium (BSM) (Himedia, Mumbai) enriched with Brucella selective supplement (Himedia, Mumbai) was used. Foetal stomach contents, uterine discharges, milk and hygroma fluid were taken in microcentrifuge tubes and centrifuged at 6000 rpm for 10 minutes and the pellets were suspended in small volume of BSM broth and were then inoculated on BSM agar plate for isolation. Foetal membranes, placenta and cotyledons were homogenized in pestle and mortar and homogenates were inoculated on BSM agar plate and incubated at 37 0 C under microaerophilic environment in a candle jar up to 3-5 days and checked daily for the growth of Brucella. Identification of isolated Brucella: Smears were prepared from suspected colonies and staining was done with Gram s stain August, 2017

and Brucella differential staining (Stamp s modification of Ziehl Neelsen) method. The smears were examined under oil immersion lens. The isolates revealed Gram-negative, small red coccobacilli with blue background in MZN staining. The standard biochemical tests (catalase, oxidase, nitrate, urease, indole and H 2 S production) were performed for confirmation of suspected colonies as per Quinn et al (1994). Confirmation of isolates by polymerase chain reaction (PCR): Suspected isolates were confirmed by PCR using Brucella genus specific primers B4/B5 (Baily et al 1992) and F4/R2 (Romero et al 1995). Sensitivity of Brucella abortus to antimicrobial agents: Common antimicrobial agents (Himedia) viz Doxycyclin, Oxytetracyclin, Enrofloxacin, Penicillin, Neomycin, Amoxicillin, Ampicillin, Streptomycin, Gentamicin and Erythromycin were used. The plates were incubated microaerobically at 37 0 C for 3-4 days and zone of inhibition was measured for comparing sensitivity and resistance of each agent. Extraction of genomic DNA: The genomic DNA of Brucella isolates was extracted as per method of Wilson (1987) with minor modification. Briefly bacteria from broth culture were lysed and proteins were digested with proteinase K. Cell wall debris, polysaccharides and remaining proteins were removed by selective precipitation with cetyl trimethyl ammonium bromide (CTAB). The high molecular weight DNA was recovered from resulted supernatant by isopropanol precipitation. BSM broth (5 ml) was inoculated with a single colony from the BSM agar plate and incubated under microaerophillic condition at 37 0 C for 3-5 days. After this, 1.5 ml of culture was pelleted by centrifugation at 6000g for 2 minutes. The pellet was suspended in 567 µl of TE buffer and to this 30 µl of SDS (10%) and 3 µl of proteinase K (20 mg/ml, Sigma- Aldrich) were added and incubated at 37 0 C for 1 hour after thorough mixing. Cell wall debris, polysaccharides and denatured proteins were removed by selective precipitation with the addition of 120 µl of 5M NaCl and 100 µl of CTAB/NaCl solution. The solution was mixed thoroughly and incubated for 10 minutes at 65 0 C. Equal volume of phenol: choloroform: isoamyl alcohol (25:24:1) was added to the tube, and spun for 10 minutes at 10,000 rpm. Remaining CTAB precipitate was then removed in the phenol / chloroform extraction. The aqueous, viscous supernatant was then transferred to a fresh microcentrifuge tube, leaving the interface behind. Equal volume (0.7 to 0.8 ml) of choloroform / isoamyl alcohol (24:1) was added and mixed thoroughly by inverting the centrifuge tube, and was spun for 10 minutes at 10,000 rpm to separate the upper aqueous and lower organic phases. The supernatant was then transferred to a fresh tube. Isopropanol (0.6 volume) was added to precipitate the nucleic acid. The tube was shaken back and forth until a stringy white DNA precipitate became clearly visible. The DNA was washed with 70% ethanol to remove residual CTAB and salts and centrifuged for 5 minutes at room temperature to repellet it. The supernatant was removed carefully and pellet was dried in a vacuum evaporator (Eppendorf Concentrator 5301). The pellet was re-dissolved in 100 µl TE buffer and 2 µl RNAase (10mg/ml in buffer) was added to each tube. The tubes were kept at 37 0 C in incubator for 2 hours and stored at -20 0 C for further use. Determination of integrity, concentration and purity of DNA: 10 µl of purified genomic DNA was mixed with 2 µl of 6X loading dye (Fermentas) and loaded on 1% agarose gel containg ethidium bromide solution (0.5 µg/ml). The gel was run at 5 volts/cm distance between the two electrodes in an electrophoresis unit (BioRad Model 200/2.0) containing 1X TBE buffer. The agarose gels were visualized under Gel documentation system. The integrity of DNA sample was determined by the presence or absence of smearing along the length of gel. Intact heavy molecular weight DNA remained stuck in the agarose wells even after running the gel under the influence of electric current. The concentration and purity of the extracted DNA was determined using Spectrophotometer (Nanodrop, Thermo). Detection of Brucella directly from samples by PCR: The extraction of DNA from samples (foetal stomach contents, uterine discharge, milk, vaginal mucus) was carried out as per the method described by Sambrook and Russel (2001). Confirmation of extracted DNA by PCR: PCR reactions were carried out in Thermal Cycler (Touchdown Hybrid Limited, UK). 1. Primer sequence: The PCR assay was carried out using Brucella genus specific primers B4/B5 (Baily et al 1992) and F4/ R2 (Romero et al 1995) as per the sequence given in table 1 which were obtained from Promega. 2. PCR protocol: Amplification reaction mixture was prepared in a volume of 25 µl containing 1X PCR buffer, 1.5 mm MgCl 2, 200 µm each deoxynucleoside triphosphate, 20 pmol each primer, 1 U of Taq DNA polymerase and 3 µl of template DNA (Table 2). For F4/ R2 and B4/B5 primers, the conditions of PCR are given in tables 3 and 4 respectively. Visualization of amplicons: 1% Agarose gel was prepared to resolve the PCR products. After electrophoresis, the gel was visualized under UV rays using AlphaImager 3400 HP Gel Documentation System (AlphaInnotech) to visualize the amplified products. The amplicon size and concentrations were determined by comparing the Gene Ruler 100 bp plus DNA ladder (Fermentas, USA) which was run along the samples. Results and Discussion Samples from brucellosis suspected animals: In the present study a total of 104 samples from brucellosis suspected bovines (78 Table-1: Sequence of primers used for detection of B. abortus Primers Sequence Size of amplified product Reference F4 5 - TCG AGC GCC CGC AAG GGG -3 905bp Romero et al (1995) R2 5 - AAC CAT AGT GTC TCC ACT AA -3 B4 F 5 - TGG CTC GGT TGC CAA TAT CAA- 3 223bp Baily et al (1992) B5 R 5 - CGC GCT TGC CTT TCA GGT CTG-3 Research in Environment and Life Sciences 719 August, 2017

Table-2: Brucella PCR Reaction mixture PCR components Required concentration Amount(µl) H 2 O Up to 25 µl 14.3 PCR buffer (10X) 1X 2.5 MgCl2 25 mm 1.5 mm 1.5 dntps (10 mm) 200 µm 0.5 Primers (40 pm/µl each) 20 pmol each 0.5 2 Taq (5 U/µl) 1 U 0.2 DNA template 100 ng 5.0 Total volume 25 Table-3: Brucella PCR program using F4/R2 primers Stage Step Temperature 0 C Duration No. of cycles I. Initial denaturation 95 4 min 1 II. Denaturation 95 1min 35 Annealing 56 1min Extention 72 1min III. Final extention 72 3 min 1 Table-4: Brucella PCR program using B4/B5 primers Stage Step Temperature 0 C Duration No. of cycles I. Initial denaturation 94 5 min 1 II. Denaturation 94 1min 35 Annealing 65 1min Extension 72 1min III. Final extension 72 10 min 1 Fig. 3: Confirmation of Brucella isolates by PCR employing F4/R2 primers Fig.1: Biochemical tests for Brucella abortus isolates Fig.4: Confirmation of Brucella isolates by PCR employing B4/B5 primers Fig.2: Test for H2S production Research in Environment and Life Sciences 720 Fig.5: Antibiotic sensitivity test for a Brucella abortus isolate August, 2017

Table-5: Clinical samples processed for isolation of Brucella organisms Sample Animal Age Month of Type of Isolation of No. (years) abortion sample organism 1b Cow 6 6 FSC - 2b Cow 5 7 FSC + 3b Cow 6 6 FSC - 4b Cow 6.5 6 Milk + 5b Cow 6 7 Milk _ 6b Cow 4 6.5 Milk + 7b Cow 5 7 Uterine fluid + 8b Cow 6 NA Vaginal mucus - 9b Cow 4 6.5 Uterine fluid - 10b Cow 7 7 Uterine fluid _ 11b Cow 5 6 Uterine fluid + 12b Cow 4.5 7 Uterine fluid + 13b Cow 4 NA Milk _ 14b Cow 5 7 Milk + 15b Cow 5 NA Milk _ 16b Cow 6 7 Milk - 17b Cow 6 NA Vaginal mucus _ 18b Cow 4 NA Vaginal mucus _ 19b Cow 4.5 NA Vaginal mucus _ 20b Cow 4 NA Uterine fluid _ 21b Cow 5 NA Milk _ 22b Cow 5.5 ROP Foetal membrane _ 23b Cow 4 4 Milk _ 24b Cow 3 6 Uterine fluid _ 25b Cow 4 NA Milk _ 26b Cow 4 4 Uterine fluid + 27b Cow 3 NA Milk _ 28b Cow 6 4 Uterine fluid 29b Cow 7 ROP Foetal membrane _ 30b Cow 6 NA Milk _ 31b Cow 4 NA Milk _ 32b Buffalo 5 H/O prolapse Milk _ 33b Buffalo 5 H/O prolapse Uterine fluid _ 34b Buffalo 5 7 FSC + 35b Cow 5 4 Milk _ 36b Cow 4 6 Uterine fluid _ 37b Cow 4.5 6 Milk _ 38b Cow 4 NA Milk _ 39b Cow 4 NA Vaginal mucus _ 40b Cow 5 4 Milk _ 41b Cow 4 ROP Milk _ 42b Cow 5 ROP Uterine fluid _ 43b Cow 4 NA Milk _ 44b Cow 6 5.5 Uterine fluid _ 45b Cow 4 6 Milk _ 46b Cow 5 NA Milk _ 47b Cow 6 NA Milk _ 48b Cow 5 6.5 Uterine fluid _ 49b Cow 4 NA Milk _ 50b Cow 6 7 Uterine fluid _ 51b Buffalo 4.5 6 FSC _ 52b Cow 6.5 H/O RB Milk _ 53b Cow 5 H/O RB Milk _ 54b Cow 6 H/O RB Milk _ 55b Cow 5 6.5 Uterine fluid _ 56b Buffalo 6.5 H/O RB Vaginal mucus _ 57b Cow 4 H/O prolapse Uterine fluid _ 58b Cow 5 H/O ROP Uterine fluid 59 59b Cow 6 H/O DystociaUterine fluid _ 60b Cow 6 H/O ROP Uterine fluid _ 61b Cow 7 H/O RB Vaginal mucus _ 62b Buffalo 7 H/O RB Milk _ 63b Buffalo 6.5 H/O RB Milk _ 64b Buffalo 6.5 H/O DystociaUterine fluid _ 65b Cow 5.5 NA Milk _ 66b Cow 4.5 NA Milk _ 67b Cow 6 5 FSC _ 68b Buffalo 6 H/O RB Milk _ 69b Buffalo 5 H/O RB Milk _ 70b Buffalo 4.5 NA Milk _ 71b Buffalo 5.5 H/O RB Milk _ 72b Cow 4.5 H/O DystociaUterine fluid _ 73b Cow 6.5 H/O DystociaUterine fluid _ 74b Buffalo 5 5.5 Milk _ 75b Cow 4.5 5 Milk _ 76b Cow 6.5 H/O RB Milk _ 77b Cow 5.5 H/O RB Milk _ 78b Cow 6 6.5 FSC + 79b Cow 5 H/O RB Milk _ 80b Buffalo 5 H/O RB Vaginal mucus _ 81b Cow 4 7.5 FSC + 82b Buffalo 5 H/O DystociaUterine fluid _ 83b Buffalo 4 H/O DystociaUterine fluid _ 84b Cow 5 6 FSC _ 85b Cow 4 5 FSC _ 86b Buffalo 4 4 FSC _ 87b Buffalo 6 H/O ROP Uterine fluid _ 88b Cow 4 H/O DystociaUterine fluid _ 89b Cow 5 6.5 FSC + 90b Buffalo 6 H/O RB Milk _ 91b Buffalo 5 H/O RB Milk _ 92b Buffalo 4.5 NA Milk _ 93b Buffalo 4 NA Milk _ 94b Cow 7 H/O DystociaFSC _ 95b Cow 5 H/O ROP Uterine fluid _ 96b Buffalo 5 H/O DystociaUterine fluid _ 97b Buffalo 5 H/O DystociaMilk _ 98b Buffalo 7 H/O RB Milk _ 99b Buffalo 5 7 FSC _ 100b Cow 7 H/O ProlapseUterine fluid _ 101b Cow 5 NA Vaginal mucus _ 102b Cow 4 NA Milk _ 103b Cow 5 7.5 Milk _ 104b Cow 4.5 NA Milk _ ROP = Retention of placenta, H/O RB = History of repeat breeding, FSC = Foetal stomach contents Research in Environment and Life Sciences 721 August, 2017

Table-6: Detection of Brucella abortus in clinical samples from aborted cattle and buffaloes Type of sample Cattle Buffaloes No. of animals tested No. of animals positive No. of animals tested No. of animals positive Foetal stomach contents 10 4 (40%) 4 1 (25%) Uterine fluid 24 4 (16.67%) 6 0 (0%) Milk 35 3 (8.57%) 14 0 (0%) Foetal membranes 02 0 (0%) 0 0 (0%) Vaginal mucous 07 0 (0%) 02 0 (0%) Total 78 11 (14.10%) 26 1 (3.84%) Table-7: Biochemical characteristics of Brucella isolates Isolate No. Catalase Oxidase Urease Nitrate reduction H 2 S(TSI) Indole Lysis by bacteriophage 2b + + + + + _ + 4b + + + + + _ + 6b + + + + + 7b + + + + + 11b + + + + + 12b + + + + + _ + 14b + + + + + _ + 26b + + + + + _ + 34b + + + + + 78b + + + + + 81b + + + + + _ + 89b + + + + + _ + Table-8: Brucella isolates found to be positive by PCR Isolate No. 2b + 4b + 6b + 7b + 11b + 12b + 14b + 26b + 34b + 78b + 81b + 89b + PCR results from cattle and 26 from buffaloes) were analyzed (Table 5). Isolation of Brucella organisms: Out of the total 104 samples, 12 were found to be positive for Brucella abortus by isolation. The isolates were confirmed to be Brucella organisms by PCR in our lab as well as at IVRI, Izatnagar. Prevalence of Brucella abortus in aborted cattle and buffaloes: Out of a total of 104 samples (78 from cattle and 26 from buffaloes) which included foetal stomach contents, uterine fluid, milk, foetal membrane, and vaginal mucous 12 (11.53%) samples {11 (14.102%) samples from cattle and 1 (3.84%) sample from buffalo} were found to be positive for B. abortus through primary isolation procedure (Table 6). In case of cattle, 4 out of 10 (40%) foetal stomach contents, 4 out of 24 (16.67%) uterine fluid, and 3 out of 35 (8.57%) milk samples were positive by isolation but no Brucella could be isolated from 2 foetal membranes and 7 samples of vaginal mucous through standard primary isolation procedure. In case of buffalo, 1 out of 4 (3.84%) foetal stomach contents was positive by primary isolation. No Brucella could be isolated from 6 samples of uterine fluid, 14 milk samples, and 2 vaginal mucous samples, respectively. The disease prevalence in Punjab has earlier been reported to be higher in cattle (20.67%) compared to buffaloes (16.41%) by using milk-elisa (Aulakh et al 2008). A possible reason for this difference could be greater exposure of buffaloes to dirty waters in routine course. Sharma et al (2003) reported the prevalence rate among cattle and buffaloes in Uttaranchal to be 28.57% and 11.53%, respectively. Identification and biochemical characterization of Brucella isolates: Identification of Brucella isolates was done on the basis of cultural and staining characteristics and biochemical parameters. Morphological characterstics like smooth, glistening, translucent and pinpoint colonies appearing after 3-5 days of incubation in a microaerophilic environment were indicative of Brucella. The colonies were found to be gram negative. The organisms appeared as coccobacillary rods whereas by Modified Ziehl-Neelson (MZN) staining they appeared to be red coccobacilli against a blue background. The colonies were subjected to biochemical tests like H 2 S production, catalase, oxidase, urease, nitrate reduction and indole test. The organisms were found to be positive for catalase, oxidase and urease and H 2 S was produced by 7 isolates. All the 12 of our isolates were positive for nitrate reduction and negative for indole test (Table 7, Figs 1 & 2). Buyukcangaz and Sen (2007) have reported that in their study, all the 8 isolates of Brucella were positive for catalase, oxidase, and urease and 7 out of 8 isolates were positive for H 2 S production and required CO 2 for isolation. Shome et al (1999) had reported Research in Environment and Life Sciences 722 August, 2017

Table-9: Susceptibility of Brucella abortus isolates to different antimicrobial agents Antimicrobial agents No. of Total no. of Percentage of Total no. of Percentage of isolates sensitive isolates sensitive isolates resistant isolates resistant isolates Chloramphenicol 12 10 83.33 2 16.66 Oxytetracycline 12 8 66.66 4 33.33 Ampicillin 12 8 66.66 4 33.33 Streptomycin 12 8 66.66 4 33.33 Gatifloxacin 12 6 50 6 50 Ceftaxime 12 6 50 6 50 Erythromycin 12 3 25 9 75 Cephalothin 12 2 16.66 10 83.33 Cotrimazole 12 2 16.66 10 83.33 Penicillin 12 0 0 12 100 that one out of three of their Brucella isolates was negative for H 2 S production. Verma et al (2000) have also observed B. abortus isolates to be negative for H 2 S and indole production. Confirmation of Brucella isolates by PCR DNA extraction from Brucella: The genomic DNA of Brucella abortus field isolates and the standard vaccine strain (S-19) was extracted by using standard bacterial DNA extraction method described by Wilson (1987). The OD 260/280 value for isolated DNA was ~1.8-1.9 for all samples indicating the purity of DNA. Confirmation by PCR: The DNA extracted from Brucella abortus isolates and S-19 when subjected to PCR using Brucella specific primers B4/B5 and F4/R2 revealed the desired amplicons at 223 bp and 905 bp respectively (Table 8, Figs. 3 & 4). Sensitivity of Brucella abortus isolates to antimicrobial agents: Brucella abortus organisms were found to be sensitive to chloramphenicol, oxytetracyclin, ampicillin, streptomycin, gatifloxacin and ceftaxime but were resistant to penicillin, cotrimazole, cephalothin and erythromycin (Table 9, Fig 5). Sensitivity of the organisms was 83.33 per cent for chloramphenicol, 66.66 per cent for oxytetracyclin, ampicillin, and streptomycin, 50 per cent for gatifloxacin, ceftaxime, 25 per cent for erythromycin, and 16.66 per cent for cephalothin and cotrimazole. The organisms were 100 per cent resistant to penicillin. The trends of sensitivity to antibiotics vary with time and abuse or overuse of certain antibiotics. Increasing resistance to newer antibiotics is an alarming trend and reflects indiscriminate use of certain antibiotics. Jeyaprakash et al (1999) had also found B. abortus to be resistant to penicillin but highly sensitive to chloramphenicol, streptomycin and cotrimazole. The existence of antimicrobial resistant Brucella abortus among cattle and buffaloes in the field is alarming since Brucellosis affected animals remain carrier for the rest of their lives and no other effective therapy is available currently. The available S-19 vaccine is not fully effective since we have come across infection in animals which had been vaccinated during calfhood (Mohan et al., 2016). The present study has revealed the existence of antimicrobial resistant Brucella abortus among cattle and buffaloes in the field in Punjab state. 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