In Ethiopia, many donkeys and horses have died because of the outbreak of a specific virus whose roots have yet to be known. With a careful diagnostic study and experimentations using different significant samples, the reasons and notion underlying such death causing the decreased population of donkeys have been investigated. Mono layer of RK 13 cells which were derived from the kidney of a rabbit were used in the experimentation at both aerobic and anaerobic process. After several hours of observation and experimentation, it is evident that there is a significant decrease on the number of cells in the infected RK13 cells. It has also the presence of cytopathic effects which are indicative of the presence of a viral agent. The presence of both acyclovir and ether has also been significant in the process of doing the experimentation. After all the procedures, the presence of Equine Herpes Virus has been pinpointed as the cause of the outbreak in Ethiopia.
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The use of donkeys, which belong to the family of horses, has been regarded as one of the most significant sources of income in the African continent. However, one major event damaged this which was the outbreak of diseases in Ethiopia which increases the rate of mortality and abortion in young foals. Abortion rate showed an overwhelming increase to 90% from 10%. Because of this, the authorities have been alarmed by this threat and they ventured into different studies and measures to remedy the continually decreasing population of foals. The mortality of the donkeys and horses in Ethiopia, according to successfully concluded studies, happened eighteen months after Ethiopia was able to receive the donkey modules from the United Kingdom and the United States of America. According to Allen and Bryans (1986), this virus was first recognized to have affected the donkeys in Kentucky in 1932. It has been recognized that different kinds of herpes viruses such as Asenine herpes virus (AHV-B) and Equine herpes virus (EHV-1) were responsible for these deaths.
The primary aim of the researcher in carrying out this study is to be able to identify and investigate on what bacteria or virus can the mortality of donkeys be attributed along with identifying the pathogenic source of this kind of disease.
The samples which will be used to be able to carry out the purpose of this study will include homogenate which is made of placenta of aborted foetal donkeys. This will include monolayer of RK 13 cells which were used to recover from the virus. The cells which will be user are those which were four wells out of eight tissues and will be incubated for 24 hours.
In this process, the RK 13 cells infected with native virus, RK 13 cells infected with ether treated virus, and RK 13 cells infected with native virus in the presence of acyclovir were used to be able to potentially identify the different properties which are present in the virus and to be also able to determine if there is a presence of various inclusion bodies and changes which might occur in the morphology of the host cell.
The first activities which will be done to conduct the study are the separation of the non-viral components from homogenate and inoculate with filtered placental homogenate. After this, the cells will be incubated at 37 degrees Celsius which shall be done in an interval of 24 hours until it reaches the end of the 96th hour. After this, an observation on the microscope under low or medium light will be done next as the samples were placed on the microscopic slides for further experimentation.
The next activity related to the conduct of the study would be the use of a fluorescent microscope to be able to identify what is the viral nucleic acid. 2.5 percent of glutaraldehyde will be used to treat RK 13 virus infected with native virus for five minutes then it will be followed with fifteen minutes staining of the sample in 0.5 ml 5mg/l acridine orange solution. Fifty percent of glycerol will also be sued and applied on the slides and they will be observed under the fluorescent microscope to be able to arrive at significant findings. However, the tow other samples which will be used for the purpose of experimentation which include the RK 13 cells infected with ether treated virus and RK 13 cells which were present with acyclovir will also be stained for a duration between thirty to forty minutes as the mentioned samples are stained using 1/20 Giemsa. Low/medium light under the fluorescent microscope will also be used to arrive at the conclusion.
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RESULTS OF THE EXPERIMENTATON
In both plates which were incubated aerobically and anaerobically, it has been found out through experimentation that there was no growth of any microorganisms.
Viral Agents and Morphology
After a series of four incubation periods which was done after every 24 hours to the sample, it is evident that there is a significant decrease with the number of cells in the virus infected cells. At the end of the 48th hour, it is also significant that the sample showed and appearance which happens to be large and swollen making the cytopathic effects more visible to the experimentation. After the 72n hour, however, it is evident among the samples that the nucleus is significantly increasing in terms of its physical size. And at the 96th hour, some cells showed multinucleated syncytium which made the sample relatively different from each other. After being exposed to the presence of two different anti-bacterial agents, it is noticed that there is an evident difference between the cytopathic effects which appeared. For the RK13 cell which was treated with ether, it is observed that there is no evident of cytopathic effect and they were observed to have the presence of some spots in the samples which appear to be dark. However, the RK13 cells which has the presence of acyclovir there were changes in morphology, it showed similarities to the native cell, and there was an observance of cytopathic effect.
After the study has been conducted and several methods of experimentation has been executed to carry out a significant finding, it can be safely assumed that the aborted donkeys showed the presence of the virus in the placenta. Hwoever, the pathogen which was isolated also proved the susceptibility of the samples to various anti-viral agents. According to Rollinson and White (1983), the acyclovir which was used in the experimentation is one anti-herpes agent which inhibits viral DNA polymerase and viral DNA replication while Coohill et al (1980) assumes that the ether used in the experimentation is an effective virucidal agent which can enable to affect the various components of the viral envelope. After having the samples observed on both ether and acyclovir, it is assumed that the presence of Equine Rhinopneumonitis (ER) is the one which is responsible for the abortion of the donkeys and the increased occurrence of different diseases associated to the respiratory system of the donkeys. EHV-1, one of the distinct viruses, is concluded to be the cause behind such outbreak in the donkeys and horses in Ethiopia.
The presence of EHV-1 is said to be responsible for various health conditions such as diseases on the respiratory system, neurological diseases, abortion, and the death of the newborn foals ââ‚¬" an event which are all evident on what happened to Ethiopia. Aside from affecting the new born, EHV-1 is also assumed to be the cause of the deaths of adult horses (Crabb, B.S. and Studdert, M.J., 1993). Hartley and Dixon (1929) illustrated that if the EHV-1 will be present during the late stages of the pregnancy of the donkeys or horses, the new born will be dead. However, it can also be alive but will only survive through few hours. Another point which must be discussed is teh fact that the modules which has been assumed to cause the outbreak were from the U.S. and U.K which widens the possibility of the fact that the donkeys on that places can be reservoirs of the said virus and these virus could have been activated because of activities such as traveling (Sutton et al, 1998).