Isolation, Biochemical Characterization, Antibiogram Pattern and PCR Based Confirmation of Brucella from Cows and Buffaloes

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Isolation, Biochemical Characterization, Antibiogram Pattern and PCR Based Confirmation of Brucella from Cows and Buffaloes
Ritesh N. Patel * , Ashish Roy, Bharat B Bhanderi, Dhaval H Vagheshwari IntroductIon T he term brucellosis is applied to a group of closely related infectious diseases, all caused by Gram-negative bacterial pathogens from the genus Brucella. Phenotypic characteristics, antigenic variation, and prevalence of infection in different animal hosts have resulted in the initial recognition of six species: Brucell (B.) melitensis, B. suis, B. abortus, B. canis, B. ovis and B. neotomae (Vizcaino et al., 2004). In addition, in the 1990s, new Brucellae have been isolated from marine mammals, and a new species, Brucella marins was proposed (Nymo et al., 2011). Manifestations of the disease may range from abortion in the cow to orchitis or epididymitis in the bull (Dougherty et al., 2013). This disease is transmitted by direct or indirect contact with infected excreta. The most important routes of transmission are the oral and venereal ones.
The economic importance of brucellosis requires the use of sensitive and rapid diagnostic methods. At present, the diagnosis of brucellosis in live dairy cattle involves either the isolation of Brucella from various samples like milk, placenta, cotyledons, and fetal stomach contents or samples the detection of anti-Brucella antibodies in milk (Hamdy et al., 2002). Recently, polymerase chain reaction (PCR)based detection of organisms has been found to be more convenient as compared to cultural isolation. PCR is an option for diagnosis of brucellosis. AMOS (from the initial letters of abortus, melitensis, ovis, and suis) PCR assay can identify B. abortus, B. suis, B. melitensis, B. ovis (Bricker et al., 2003). The present study was carried out on isolation, antibiogram, and PCR based identification of Brucella species from samples of reproductive disorders in cows and buffaloes.

MAterIAls A n d Methods
A total of 114 samples comprised of vaginal swabs, aborted material, milk, and placenta were aseptically collected from cows (98) and buffaloes (16) in and around Anand district.

Bacterial Culture
Isolation of Brucella organisms from the vaginal swabs, aborted material, milk, and placenta from cow and buffalo was carried out after collection in transport swab (Hi-media transport swab w/Amies medium w/o charcoal in polystyrene tube). Each swab collected from an animal was separately streaked on Brucella agar medium (BAM) plates in duplicates. One plate was incubated aerobically in an incubator at 37 o C (without CO 2 ), and the other incubated at 37 o C aerobically in an atmosphere of 5% CO 2 in a CO 2 incubator for minimum 15 days. The plates were observed every 24 hours for the growth. The suspected colonies of Brucella were picked up and transferred to another BAM plates and incubated under 5% CO 2 tension to obtain a pure culture.

Identification of Brucella Isolates
Cultural and biochemical tests like oxidase, catalase production, nitrate reduction, urease, indole, VP, H2S production, motility test, and agglutination reaction with anti-B. abortus serum were carried out for the identification of Brucella isolates (Parlak et al., 2013).

Reference Bacterial Strain
The vaccine strain Brucella abortus cotton strain19 (IIL, Hyderabad, India) was used as reference bacterial stain for cultural and molecular work.

Bacterial DNA Extraction
Suspected colonies from BAM plates were streaked on BAM slants. Slants were incubated at 37°C for 4 to 5 days at 5% CO 2 tension. Bacterial colonies were picked and suspended in 100 μl DNAase free mili Q water. The colonies were boiled for 15 min, cell debris were removed by centrifugation, and 3 μl of the supernatant was used as a template. Polymerase Chain Reaction: The extracted DNA preparations were screened with genus-specific Brucella PCR using B4/B5, JPF/JPR and F4/R2 primers (Table 1). For all PCR reactions 3µl (90 ng) template DNA was taken and added to the reaction  (1994). The Bruce-ladder PCR was carried out as per methods described by Garcia-Yoldi et al. (2006). The PCR product was run on a 1.5 % agarose gel along with DNA ladder for 90 min at 105 V, stained with ethidium bromide (1 mg/ mL), and visualized under UV light using a gel documentation system.

results A n d dIscussIon
Out of 114 samples processed from vaginal swabs, aborted materials and milk, three (all from cows) isolates were recovered on BAM and were presumed to be of Brucella. All, three isolates of Brucella were from abortion cases in cows. All three isolates (named as C1, C2, and C3) were Gram-negative and MZN positive coccobacilli. Biochemical tests showed that these isolates were positive for oxidase, catalase production and nitrate reduction. While negative for urease, indole, VP, H 2 S production, and motility. All the isolates showed agglutination with Brucella-mono specific antiserum. Based on cultural and biochemical tests, the isolates were confirmed to be B. abortus. The overall prevalence of Brucella infection, as detected in the present study by cultural isolation, was 2.63% (03/114). Pal and Jain (1985)  In the present study, all the isolates of Brucella were found to be 100% sensitive to Streptomycin, Tetracycline, Amikacin, Erythromycin, Pefloxacin, Amoxyclav, Spectinomycin, and Norfloxacin. At the same time, all the isolates were found resistant to Ampicillin. Ghodasara et al. (2012) reported that all the Brucella isolates were sensitive to all the antibiotics tested.
JPF/JPR primer pair generated a 193bp (Fig. 1) amplicon from reference strains as well as from two isolates of Brucella but failed in C2. Leal-Klevezas et al. (1995) also used this primer homologous to regions of the gene coding for an omp2 for the detection of Brucella in blood and milk of the infected animals and obtained promising results. Navarro et al. (2002) and Kanani et al. (2008) also used the same primer for the detection of Brucella in infected human blood and bull semen. Patel et al. (2008) carried out PCR based detection for Brucella organisms in 53 milk samples collected from normal milch cattle by Brucella genus-specific primer pairs, and one isolate was positive by JPF/JPR primer pair.
B4/B5 primer pair generated a 223bp (Fig.2) amplicon size from reference strains as well as, all the three isolates presumed to be Brucella. This bcsp31 gene based primer has also been successfully used by Kanani et al. (2008) for detection of Brucella DNA bull semen. Similar results were also reported by Morata et al. (2001), Navarro et al. (2002) and Boeri et al. (2018) using same primer pair for diagnosis of brucellosis.
F4/R2 primer pair generated a 905bp (Fig. 3) from reference strains as well as all the three isolates of Brucella. Romero et al. (1995) applied this primer pair to DNA extracted from all of the representative strains of the species, biovars of Brucella and from 23 different Brucella isolates and amplified 905bp fragment. Similar amplicon size were obtained from milk and lymph tissues by Kanani et al.(2008) from bull semen and Patel et al.(2008) from milk by using same primer.
AMOS PCR assay is a multiplex primer assay that uses a five-primer cocktail. One primer anneals to the IS711 element. As designed, B. abortus amplifies a 498 bp product, B. melitensis amplifies a 731 bp product, B. ovis amplifies 976 bp product and B. suis amplifies a 285 bp product. AMOS PCR assay was developed to differentiate between field strains, vaccine strain S19 and RB51. The product size of 498bp was amplified for all the three isolate as well as B. abortus cotton strain 19 using AMOS primers cocktail indicating our isolates belonging to B. abortus species (Fig. 4). Similarly, Matope et al. (2009)  In the present study, all the three isolates as well as the reference B. abortus cotton strain 19 could amplify products of 1682bp, 794bp, 587bp, 450bp and 152bp using cocktail of 8 pairs of primer pair and the product size were specific for B. abortus using Bruce ladder (multiplex) PCR technique (Fig. 5). Results showed that microbiological typing and multiplex Bruce-ladder amplification were identical for all Brucella isolates tested. In a similar study, Lopes et al. (2014) found that all field strains identified to the species level by biochemical and physiological tests were confirmed by the genus-specific PCR and by the Bruce-Ladder PCR. None of the field strains of B. abortus presented the profile expected for vaccine strains S19 and RB51.

conclusIon
Among the three different genus-specific primer pairs used (B4/B5, F4/R2 and JPF/JPR) for identification of Brucella organisms, B4/B5 and F4/R2 primer pairs were found to be more sensitive for identification of Brucella organisms. For the species identification, multiplex PCR named AMOS PCR and Bruce-ladder could identify B. abortus.

AcknowledgeMents
Authors thank the authorities of Anand Agricultural University, Anand and Dean of the Veterinary Faculty for the facilities provided for this work.