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Using PCR to Diagnose Brucella Abortus in Aberdeen Angus

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 speci?city (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 identi?cation 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 ?M
each), Taq DNA polymerase, 1ng of extracted DNA of Angus cattle with suspected Brucella abortus, Sterile distilled water (to total 50 ?L
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:
Add 5 ?L of the PCR buffers (50 mM KCl, 10 mM Tris-HCl, pH 9.0),

Add 3 ?L of 1.5 mM MgCl2,

Add 5 ?L of 200 mM (each) for the four nucleotides (dNTPs),

Add 1 ?L of 1.5 U Taq DNA polymerase.

Add 1 ?L of the primers

Add 1ng of the extracted DNA
Add sterile water to bring the final volume to 50 ?L .

Tap or centrifuge briefly to bring the content to the bottom.
Place in the PCR machine for thermal cycling.
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.

REFERENCES
Al Dahouk, S., Le Flèche, P., Nöckler, K., Jacques, I., Grayon, M., Scholz, H. C., …

Underestimated Plasmodium Malariae

Over the past decade, malaria control strategies in Africa have decreased in numbers of malaria cases and deaths (Lo et al., 2017). Nevertheless, Plasmodium falciparum malaria is still a major case of elimination. The malaria elimination programs are focused on this parasite, which has been resourced to P.vivax. There are two other malaria parasites, which don’t receive much attention, P.malariae and P.olvale. These are the most neglected tropical diseases (Lo et al., 2017). The following paper will be discussing on the article Plasmodium malariae Prevalence and csp Gene Diversity, Kenya published in 2017 that will explain the findings of how and why P. marlariae should be considered in the category of malaria elimination in Africa, with the need of sensitive outreach applicable methods to specifically identify P.malariae in malaria endemic regions.
Malaria and Africa
Between the months of June and August in the year of 2014 and 2015, blood samples were collected from individuals in 4 villages in Western Kenya. These areas were tested due to the high and stable rates of malaria transmission among the ages of 5-14 years of age (Lo et al., 2017). The writers also collected community samples in 7 public schools between the ages of 6 to 15 years to determine the difference in the adult population (Lo et al., 2017). They examined a total of 663 samples from the selected regions, which had given them an estimation in parasitic prevalence, having the individuals with no exhibiting fever or malaria like symptoms (Lo et al., 2017).
They had a second group of blood samples that were clinical samples collected from 113 male and 132 female patients less than 1 to 76 years of age in three selected hospitals. These patients had fever or malaria like symptoms that were resulting in a high percentage of being positive for Plasmodium spp. by microscopy (Lo et al., 2017).
As a result of the 663 samples of asymptomatic individuals, 5.3% were detected for P.malariae. From this total, 29 were mixed with P.falciparum infections and 6 were found with P.malariae monoinfections (Lo et al., 2017). P.malariae was found to be the most common in Kombewa and Kendu Bay in younger individuals less than 15 years of age. Among the of symptomatic patients, a high percentage of P.malariae were in infants or very young children of less than 5 years of age (Lo et al., 2017).
The team found that P.malariae is most common among infants and young children than adults. Senegal, West Africa had 91% cases of clinical P.malariae that occurred in children between the ages of 5 to 9 years (Lo et al., 2017). Although they found that children are most vulnerable to the parasite P.malariae, it is not associated strictly with age. The writers expressed, “Chronic nephrotic syndromes attributed to P.malariae have been reported and shown to be associated with significant illness from anemia in young children.” Further investigations have to be made due to the lack of their hematologic data that they have obtained.
In Africa, the standard medication to treat for P.malariae monoinfection is Cholorquine, which is an anti-parasite and immunosuppressive drug. P. malariae increases the development of P. falciparum gametocytes, which can endure without proper antimalarial treatment. The morphology of P.malariae and P. falciparum ring form are very similar to each. (Lo et al., 2017). They emphasize the need for sensitive methods of improvement to specifically diagnose P. malariae and provide systemic epidemiologic data in order to avoid any misdiagnosis.
Plasmodium Malariae
Malaria is gram-negative severe and sometimes-fatal disease caused by parasitic protozoa that infects anopheles mosquitos, which feeds on humans. There are four different species that cause malaria, which are P. ovale, P.vivax, P.malariae, and P. falciparum, being the most dangerous for humans. In the plasmodium life cycle, the female infected anophetes mosquito injects sporozoites into the host’s blood stream. (Bauman, 2018). These sporozoites travel quickly into the host’s bloodstream invading their liver cells and undergoing a process known as schizogony.
Approximately two weeks from the bite, the liver cells rupture and release about 40,000 merozoites into the blood, which completely damages the host’s liver (Bauman, 2018). These merozoites then penetrate through the host’s erythrocytes, where they become a trophozoite showing in its ring (Bauman, 2018). During this invasion, merozoites are released and break through the host’s erythrocytes, which occur every 48 to 72 hours depending on the type of Plasmodium. Lastly, they are released into the body taking over the host’s immune system.
Morphology
The morphology of P.malariae’s rings has a sturdy cytoplasm and a large chromatin dot. Their trophozoites have compact cytoplasm and a large chromatin; some brown pigments that can be seen through observation in the microscope (CDC, 2018). P. malariae’s schizonts have 6 to 12 merozoites with large nuclei being clustered around a mass with brown pigments, with their gametocytes shapes are oval to round with scattered brown pigmentation (CDC, 2018).
There are approximately 5,000 genes that encode for traits in malaria that assist in pathogenicity (CDC, 2018). The virulence factors associated with P.malariae include malaria secretome injecting toxins into the body changing the body chemistry, having the mosquitos detect the host’s sweet blood, to attract more. The reproductive cycle hides the parasite and merozoites form within the host’s vesicles affecting the immune system’s ability for detection (Bauman, 2018). There are certain traits that increase the resistance to this disease such as the presence of the sickle-cell gene, lack of Duffy antigen on erythrocytes, and the presence of two genes for hemoglobin C (Bauman, 2018).
Growth Conditions
According to the Centers for Disease Control and Prevention, malaria is found depending on the climatic factors such as humidity, temperature, and rainfall. This parasite is transmitted in tropical and subtropical areas where the anopheles mosquitos can survive and duplicate (CDC, 2018). This parasitic protozoon causes more than 1 million deaths yearly. Malaria does not occur everywhere around the world, it is more intense in warmer regions closer to the equator, where this parasite is transmitted year-round. This mainly occurs in Africa South of the Sahara and some parts of Oceania, where New Guinea and Papua is located, where P. falciparum is transmitted most (CDC, 2018). In cooler climates such as Europe and the United States P.vivax is more prevalent because of the tolerance of lower ambient temperatures (CDC, 2018).
Diagnosis, Treatment, and Prevention
Although P.malariae is not as common as P.falciparum, it should still be taken as serious and be recognized and treated promptly. It can be a challenge diagnosing a patient with malaria since this is not an endemic in the United States anymore. Many health care providers may forget to consider the possible chances of their patient having this disease. Technicians may oversee or lack the experience to detect the parasites when undergoing examination through blood smears under the microscope (CDC, 2018).
It is emphasized to pay close attention to the patient’s symptoms such as fever, chills, headaches, nausea, muscle aches, fatigue, anemia, and vomiting. These factors are flu like symptoms and common viral infections, but malaria should also be considered as a possible diagnosis. To identify malaria parasites, laboratorians collect droplets of blood from the infected patient to undergo a blood smear on a microscope and are stained using the Giemsa stain procedure to determine it’s diagnosis (CDC, 2018).
Some Plasmodium strains are resistant to antimalarial drugs, so drug resistance tests must be performed to assess the susceptibility of the parasite. The parasites are cultured in vitro tests to analyze the drug concentration that suppress the parasite growth (CDC, 2018). The increase of malaria being drug resistant poses a major threat in the increase statistics of morbidity and mortality. According to the Center for Disease Control and Prevention, it is unknown if P.malariae is resistant to any antimalarial drugs as of yet (CDC, 2018). Malaria can be severe and increase the risk of becoming a fatal disease. Treatment should be considered and initiated immediately. There are many antimalarial drugs, and most are active to fight against the parasite, such as chloroquine, quinine, doxycycline and atovaquone-proguanil (CDC, 2018).
It is important to help prevent this disease from spreading, especially in developing endemic countries such as Africa. Educating people on this parasite and controlling the Anopheles mosquitos from entering in their homes by providing bed side nets and insect repellent containing DEET. For those who travel outside of the United States, it is crucial to take antimalarial dugs to prevent the signs and symptoms of malaria. Currently, there is no antimalarial vaccination approved on the market, but Bill and Melinda Gates Foundation have developed a vaccine that has so far been accurate on 15 subjects that were tested on (CDC, 2018). Giving hope, this vaccination will soon be approved and decrease the morbidity and mortality of this disease.
Conclusion
According to the article, malaria still remains a major public health concern in countries, where this disease is transmitted most often. Specifically, young children, pregnant women, and immune compromised patients are at a higher risk for malaria. There are many malaria-controlled strategies that are in affect, but most are not affordable in all regions. P.malariae is very crucial to analyze through the microscope because of the similarity of P.falciparum, which is usually overlooked at many times, underestimated and misdiagnosed. The more education there is on the prevention of this disease, the less endemic cases there will be around the higher risk regions. It is crucial to take this into account on this local epidemiological case of malaria and the levels of resources that should be available for the regions that are at higher risk.
Reference
Bauman, Roberts. (2018). Microbiology with diseases by Body Systems. Pearson Education, Inc. (Print)
CDC – Malaria – About Malaria. (2018, March 29). Retrieved from https://www.cdc.gov/malaria/about/
Lo, E., Nguyen, K., Nguyen, J., Hemming-Scroeder, E., Xu, J., Etemesi, H., … Githeko, A. (2017). Plasmodium malariae prevalence and csp gene diversity, kenya, 2014 and 2015. doi:10.3201/eid2304.161245

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