Volume 6, Issue 5, October Issue - 2018, Pages:808-815
|Authors: K. M. Chandrashekar, Shrikrishna Isloor, D. Rathnamma, B.M. Veeregowda, Raveendra Hegde, R. Sharada, G. S. Naveen Kumar, Nagendra R. Hegde|
|Abstract: The present study explores the application of conventional PCR and Mole v/s Avogadro’s number using single copy genes of Staphylococcus aureus and Escherichia coli for assessing bacterial load in milk samples from mastitis cases. The nuc and uidA genes produced the lowest detection limit for Staph. aureus and E. coli respectively. The present findings estimated that, as few as 60 and 89 genome copies or organisms of Staph. aureus and E. coli were detected, respectively. Based on the LOD of molecules, standard graphs for these genes were generated and this knowledge was applied to milk samples from the field. Spiking known amount of genomic DNA (which in turn indicates organisms based on Avogadro’s number) in LB broth and pasteurized milk was carried out to compare the input with output and simulating field conditions. A total of 90 samples from subclinical cases of mastitis were collected from four organized farms located at various villages. Out of 90 samples, 29 (32.22 %) were showed culture and duplex PCR positive. Of these, 12 (13.33 %) were found to be positive for E. coli alone, nine (10 %) were found to be positive for Staph. aureus alone, and eight (8.88 %) carried a mixed infection for both. In conclusion, this study suggests that quantification of bacteria directly from milk by the conventional PCR can be an alternative to time consuming conventional culture method and expensive real-time PCR, but requires extensive standardization.|
|Full Text: 1 Introduction Mastitis continues to be the most important economical in modern dairy industry affecting the quality of milk, milk production, farm economics, and animal welfare. Subclinical mastitis (SCM) is considered as the main form of mastitis in modern dairy herds, affecting 20 per cent to 50 per cent of cows in given herds (Pitkala et al., 2004; Birhanu et al., 2017). In bovines, disease is most frequently caused by microbial infection, commonly associated with Staphylococcus aureus (Staph. aureus) and Escherichia coli (E. coli) which are causative agents of contagious and environmental mastitis, respectively. Detection of SCM is based on conventional tests like Somatic cell count (SCC), California mastitis test (CMT), Electrical conductivity (EC) and Bromothymol blue test (BTB) Whereas, these tests are qualitative, indirectly reflect the infection without exact etiological agent and complicated to interpret, and do not truly indicate the severity of infection (Preethirani et al., 2015). Estimation of the bacterial load could enable us to understand the severity of infection. Currently employed techniques for quantification of bacteria are plate count method and most-probable-number (MPN) method (Pascual & Calderon 2000). The disadvantage of plate count technique is negative culture (Phuektes et al., 2001) and time-consuming (Hogan et al., 1999) whereas MPN is also laborious and time-consuming (Pascual & Calderon 2000). Considering the limitations with special reference to longer time, manpower, and cost ineffectiveness associated with the above mentioned techniques, the use of DNA-based assays has become popular recently. The Polymerase Chain Reaction (PCR), which is highly sensitive, specific and so far have been reported only for the qualitative detection or identification of bacteria at earlier stages of infection, but application to gauge the bacterial load which inturn indicates the severity of the infection are lacking. However, real-time PCR can be used to quantify bacterial DNA and thus has potential for accurate enumeration of microorganisms, but high cost is a limiting factor for its wide applicability. In the present study, the concept of Mole v/s Avogadro’s number is explored for quantitative estimation of Staph. aureus and E. coli in bovine subclinical milk samples by conventional PCR assay. Avogadro’s number is the number of elementary units such as atoms, molecules or ions in one mole of a substance. Here single copy genes, nuc for Staph. aureus and uidA for E. coli were selected based on its minimum detection limit as mentioned by Chandrashekar et al. (2015). 2 Materials and Methods 2.1 Standardization of duplex PCR 2.1.1 Strains and genomic DNA Reference cultures of Staph. aureus (Accession No. JN247783) (MTCC No. 96) and E. coli (Accession No. JF926686), were obtained from Microbial Type Culture Collection, Chandigarh, India. The genomic DNA from reference strains were extracted from Staph. aureus and E. coli bacteria using the AMpurE Bacterial gDNA Mini Spin kit (Amnion Biosciences Pvt. Ltd., Bangalore), as per the manufacturer’s recommendations. Whereas, genomic DNA from the neat and diluted field milk samples were extracted by using Cremonesi method (Cremonesi et al., 2006). The purity and concentration of the genomic DNA was estimated by using nanodrop spectrophotometer (Thermo Scientific, U.S.A.) 2.1.2 Duplex PCR Five µl of the extracted genomic DNA from the standard reference strains were subjected to PCR containing 2.5 µl of 10X Taq Pol Assay Buffer, 0.5 µl (3 U / µl) of Taq Polymerase, 1.0 µl (10 mM / µl) of dNTP mix (i.e., 2.5 mM of each of the four dNTPs), 0.5 µl (10 picomoles / µl) of each of the forward and reverse primers, in a final volume of 25 µl (Chern et al., 2011; Hegde et al., 2013). The primers and cycling conditions are detailed in Table 1. Amplification was performed for 30 cycles in all cases. The amplified products (15 µl) were subjected to electrophoresis in 2% agarose gel stained with ethidium bromide, and visualized and documented by using automatic gel doc system (Gel Doc XR; Bio-Rad., U.S.A). The above procedure was applied for the genomic DNA extracted from field milk samples. 2.2 Sample collection A total of ninety milk samples were collected from four organized farms (Farm A, B, C and D) and various villages (Doddamandiganahalli, Bittagowdanahalli, Kudrugundi, Aladahalli, Rachenahalli, Doddapura, Cheeranahalli, Guddenahalli) in and around Hassan based on CMT. Milk samples were collected in 10 mL sterile tubes following strict aseptic measures and immediately transported to the laboratory in refrigerated condition and processed for SCC. Forty samples which having high SCC (> 5,00,000 cells / mL of milk) (Table 2) were log diluted in LB broth, 100 µl from each dilution was plated on MSA (Staph. aureus) and MCA (E. coli) and genomic DNA was extracted from the rest of the remaining dilutions by Cremonesi method and quantified as above (Cremonesi et al., 2006). Duplex PCR for single copy genes of Staph. aureus (nuc) and E. coli (uidA) was carried out. Quantification of bacterial load in the milk samples were estimated based on the concentration, amplicon size and size of the genomic DNA (assuming Staph. aureus and E. coli to contain 2.81 Mbp and 4.68 Mbp long genomes) by using the Avogadro`s number, formula as follows (Hein et al., 2001; Taponen et al., 2009). Weight in Dalton (gram / mol) = (base pair size of DNA) X (330 Dalton / nucleotide X 2 nucleotides / base pair) |
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