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Using PCR to Diagnose Brucella Abortus in Aberdeen Angus

2686 words (11 pages) Essay in Biology

08/02/20 Biology Reference this

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Abstract

Embryonic mortality refers to the losses occurring from conception until embryonic differentiation is complete (approximately 45 days), which can be differentiated into abortions and premature deliveries. An abortion in cattle is regarded as an expulsion before a full term of a conceptus incapable of independent life. This is caused by the species of bacteria known as Brucella abortus. An effective way to diagnose the presence of the bacterium responsible for this loss is the use of Polymerase Chain Reaction (PCR). The supremacy of PCR will be demonstrated with its specificity and amplification of the bacterium in the body fluids of infected Aberdeen angus.  This will give reliable, and accurate detection of the Brucella abortus. This project seeks to outline the protocols required for the diagnosis of Brucella abortus in Aberdeen angus with the use of PCR

 

Keywords: Embryonic mortality, bacterium, body fluids.

 

1.0              INTRODUCTION (BACKGROUND)

Reproductive failure regardless of cause and when these losses occur in the gestational duration in cattle is being referred to as Reproductive Dysgenesis (Miller, 1977). Embryonic mortality refers to losses occurring from conception until embryonic differentiation is complete (approximately 45 days) (Ayalon, 1978). Abortion and premature deliveries refer to those losses that occur during the fetal period from differentiation until parturition. The discharge before full period of a conceptus incapable of independent life is known as abortion, while a premature delivery is an expulsion before the full term of a fetus capable of independent life. Aberdeen angus cattle are naturally polled in the US and have black as the dominant color (Ibsen, 1933). They are known to be good-natured, adaptable, harsh weather resistant, undemanding, extremely early maturity and high carcass yield with nicely marbled meat. Angus female calves have been mostly used in crossbreeding because they have improved milking ability and carcass quality. They also reproduce easily, with good calf rearing ability. They are used as a genetic dehorner as it is a dominant characteristic in the polled gene (Spurlock, et al., 2014). Black Angus is now the most popular beef breed of cattle in the United States with 324,266 animals registered in 2005 (Stothard, et al., 2011).

The results of infections caused by Brucella abortus in the breed are abortions and reduced milk production level. The animals may be seen as no or little symptoms of the disease immediately the acute period of the disease is over. Chronically, the Brucella cells can be found in the mammary gland and supramammary lymphatic nodes of more than 80% of the infected animals; thus the secretion of the pathogen continues in the body fluids (Nicoletti, 2010; Godfroid and Kasbohrer, 2002; Guigon et al., 2008; Kattar et al., 2008). In the U.S., B. abortus has been reported to have been eliminated from domesticated animals but is still continued in elk and bison in the Yellowstone area (Godfroid, et al., 2005). These animals may transmit brucellosis to livestock, especially cattle grazed on open ranges (Seleem, et al., 2010).

Current diagnosis of brucellosis is based on serological and microbiological tests. Some of the well-known flaws of the serological methods are inconsistent sensitivity and specificity (Godfroid 2002, Bricker et al. 2003, Kattar et al. 2008). Furthermore, there have been continually reported to cross-react with antigens apart from those from Brucella spp. (Godfroid 2002, Mick et al., 2014). Microbiological isolation and identification have also been presumed to be the most reliable methods of diagnosing for brucellosis, but, these techniques are not always successful, as they are cumbersome, and pose a great risk of infection for the laboratory technicians (De Santis and Jerry, 2011). PCR (Kee et al., 1994) is a better and promising preference for the diagnosis of B. abortus. It has been used alone or in combination with labeled probes for the detection of B. abortus from highly contaminated aborted tissues (Chacón-Díaz et al., 2015) or isolated bacteria (Al Dahouk et al., 2007). The prevalence of brucellosis reaches as much as 200 cases per 100, 000 of the population in some regions of the world; besides, the infection has become widespread in many countries including the US (Gwida et al., 2010). There is a need for the diagnosis and control in the Angus breed to prevent its spread to humans.

 

2.0 RESEARCH GOAL

The goal of this project is to use PCR to diagnose the presence of Brucella abortus in Aberdeen angus breed in the US.

3.0  MATERIALS AND METHODS

Protocol for DNA Extraction: Heparin will be added to 3 ml of the blood sample, and the DNA will be extracted as follows. 400ul of the sample will be centrifuged at 4,000 3g for 3 min. The cell pellets will be re-suspended in 1ml of erythrocyte lysis solution (155 mM NH4Cl, 10 mM NaHCO3, 100 mM disodium EDTA [pH 7.4]), mixed, and centrifuged. Treatment with erythrocyte lysis solution will be repeated until the leukocyte pellets lost all reddish coloring. 400 ul of lysis solution (2% Triton X-100, 1% sodium dodecyl sulfate, 100 mM NaCl, 10 mM Tris-HCl [pH 8.0]) and 10 ml of proteinase K (10 mg/ml) will be added to the samples, and the contents mixed thoroughly and incubated for 30 min at 50 oC. 400ul of saturated phenol (liquid phenol containing 0.1% 8-hydroxyquinoline, saturated, and stabilized with 100 mM Tris-HCl [pH 8.0] and 0.2% 2-mercaptoethanol) will be added, and the contents mixed thoroughly and centrifuged at 8,000 3 g for 5 min. The aqueous layer will be transferred to a fresh tube, and an equal volume of chloroform isoamyl alcohol (24:1) will be added; the tubes will be mixed thoroughly and centrifuged at 8,000 3 g for 5 min. The upper layer will then be transferred to a fresh tube, and 200 ml of 7.5 M ammonium acetate will be added and mixed thoroughly. Samples will be kept on ice for 10 min and centrifuged at 8,000 3 g for 5 min, and the aqueous content transferred to a fresh tube. Two volumes of 95% ethanol will be added, the contents mixed, and the tubes will be stored at 22oC. DNA will be recovered by centrifuging the samples at 8,000 3 g for 5 min. The DNA pellets will be rinsed with 1 ml of 70% ethanol, dried, and re-suspended in 20 ml of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM disodium EDTA). The concentration will be determined after dissolving the DNA in 70% ethanol.

PCR reaction: Primers having the genes of the bacterium (Brucella abortus) will be used to diagnose its presence. If the bacterium is present, there will be annealing of the primers with the extracted DNA, confirming the presence of the bacterium in the breed. If the breed is not having the gene of Brucella abortus in its DNA, there will be no annealing with the DNA extracted.

Materials: PCR buffer (10X), dNTPs (2mM each), MgCl2 (25mM), Primers (10

 μM

each), Taq DNA polymerase, 1ng of extracted DNA of Angus cattle with suspected Brucella abortus, Sterile distilled water (to total 50

 μL

after desired amount of extracted DNA has been added).

Precautions

-          Add the component that is highest in volume. This is easier than adding the smallest volume to an empty tube.

-          Keep track of what reagent has been added to the PCR reaction

-          Reduce contamination by changing tips at each step

-          Ensure to pipette all fluid from the tip

-          If tips with a cotton plug that are aerosol resistance are available, they’ll be used.

-          To prevent degradation, primers, dNTPs, Extracted DNA and DNA polymerase should be placed on ice.

-          Ensure to use high-quality dNTPs for the PCR reaction.

Since I’ll be making use of a single PCR reaction, there’ll no need to prepare a master mix.

Protocol:  

  1. Add 5

    μL

    of the PCR buffers (50 mM KCl, 10 mM Tris-HCl, pH 9.0),

  2. Add 3

     μL

    of 1.5 mM MgCl2,

  3. Add 5

     μL

    of 200 mM (each) for the four nucleotides (dNTPs),

  4. Add 1

     μL

      of 1.5 U Taq DNA polymerase.

  5. Add 1

     μL

    of the primers

  6. Add 1ng of the extracted DNA
  7. Add sterile water to bring the final volume to 50

     μL

    .

  8. Tap or centrifuge briefly to bring the content to the bottom.
  9. Place in the PCR machine for thermal cycling.
  10. The machine is set to perform the 3 main steps of PCR, which are DNA denaturation, Primer annealing and Primer extension. DNA will be denaturated at 95 oC for 4 min; followed by primer annealing at 65 oC for 1 min, then the extension of the primers will be done at 72 oC for 1 min.

4.0  EXPECTEDRESULTS

PCR products are visible under UV light as bands. DNA is charged negatively because of its phosphate backbone. 8 ul of the amplification reaction mixture will taken and fractionated in a 1.5% agarose (or 8% polyacrylamide) gel containing 13 TBE (100 mM Tris-HCl [pH 8.0], 90 mM boric acid, 1 mM disodium EDTA), stained with an ethidium bromide solution (0.5 mg/ml), and visualized under UV light. If positive, the bands will reveal the presence of B. abortus bacterium. If the bacterium is absent, no annealing on the DNA will be found, therefore, no infection or the cause of the abortion in the cattle was not as a result of B. abortus.

5.0  CONCLUSION

In conclusion, DNA extraction and PCR procedures were useful for detecting the presence of the pathogen. The protocols presented will make detection of B. abortus by PCR easy. The amplification with the annealing of the primers will help to diagnose the presence of the pathogen in Aberdeen angus. If the pathogen is not present, there will be no annealing of the primers nor amplification through PCR. With this method, diagnosis of a pathogen will be very efficient. It will serve as an important tool for success and without reservation, an accurate and diagnosis of the bacterium. PCR through this protocol will demonstrate to be highly sensitive, dependable, specific and efficient for use in the accurate detection of B. abortus in the body fluids, such as blood. This study reached the goal of using PCR to diagnose the presence of Brucella abortus in Aberdeen angus.

 

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