6201NATSCI Prevalence Of Borrelia Spp In Ixodes Ricinus Dissertation Sample

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1.0 Introduction to Prevalence Of Borrelia Spp In Ixodes Ricinus Tick Populations Collected From Two Sites In The UK

Tick-borne diseases are among the rising diseases in the world because of its effects on both human and animals. Among them, such diseases as “Lyme borreliosis popularly known as Lyme disesea are widespread. It is an infection caused by the “Borrelia burgdorferi sensu lato group” of bacteria and spread through the ticks of the Ixodes genus. In Europe, this bacterium is mainly transmitted by the “Ixodes ricinus tick” (Hansford et al. 2022). This are found in different habitats including the woodlands and the grasslands and come into interaction with the various hosts.

Seasonal dynamics and specific agents of “Borrelia spp” differ in different geographical regions, climate, host-endemic presence or availability and environmental conditions. Examining the “levels of infection of ticks” in certain areas enables one to evaluate the presence of “Lyme disease” in those areas (Hansford et al. 2023). Such spectacular geographical regions as the “Lake District” and “Kielder Forest”are the most suitable areas in the UK for studying “Borrelia concentrations” as both these regions are endemic to ticks. If the infection rates of ticks in these areas are known, it will in the best interest of introducing the necessary precautions against the disease.

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6201NATSCI Prevalence Of Borrelia Spp In Ixodes Ricinus Dissertation Sample

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1.1 Background

Ticks are “minute haematophagous arthropods” capable of transmitting microbes such as “bacteria, viruses, and protozoa.” Unlike other vectors such as mosquitoes that feed once and inject the pathogens into the host body, ticks fix themselves to the host and feed for several hours or days, thus enhancing the chances of disease transmission. The sheep tick, scientifically referred to as “Ixodes ricinus”, is the most prevalent tick species existing in Europe. Eserves a wide range of hosts; feeds on mammals, birds, and reptiles, and thus offers these diseases a chance to jump from one species to the other.

Thus, the “life cycle of I. ricinus” is divided into three stages known as larva, nymph and adult stage. It has to feed on blood to progress to the other stage of its life cycle. To sum up, this species can be summarized as follows: These are the periods when the tick can engulf pathogen and also disseminate them (Luu et al. 2021). Nymphs are regarded as the most important for disease transmission for the same reason: Nymph is rather small and consequently it can feed to the full. “Lyme disease” is “Regional endemic zoonotic disease” which infers that the disease is transmissible from animals to the human beings. The actual pathogenic agent is a complex of the species of the bacteria: “Borrelia burgdorferi s.l’. It is estimated that in Europe only four different species belonging to this category are known to act as a vector to humans transmitting “Lyme disease”. 1) “Borrelia afzelii” (main disease with manifesting skin symptoms), 2) “Borrelia garinii” (mainly |from the neurological group, 3) “Borrelia burgdorferi sensu stricto” as the initial stage of the pathogen which mainly affects the joints and result in “Lyme arthritis”.

When a “tick bites” a human, the bacteria penetrate in the human bloodstream and give signs similar to a flu, such as “fever, fatigue, headache, muscle” aches among others (Dyczko et al. 2022). The first-known symptom is the circular skin rash known as “erythema migrans” or “bull’s eye lesion”. In its advanced stage, the disease causes inflammation of the nervous system, the joints and the heart and thus its effects are rather serious. “Ixodes ricinus” is ectothermic thus its concentration and population density vary concerning climate and other environmental factors such as temperature, humidity as well and hosts.

1.2 Research Aim and Objectives

The purpose of this paper is to determine the proportion prevalence of Borrelia spp. in Ixodes ricinus ticks collected from two different locations in the UK. This study aims at identifying prevalence levels of Borrelia spp. and whether the two places display any differences in the prevalence rates of this vector-borne pathogen and to identify factors responsible for differences in the ‘geographical distribution of tick-borne pathogens’.

The objectives of this study are as follows;

• To establish the ‘occurrence levels of Borrelia spp. in I. ricinus ticks’ collected from the two selected sites in the UK.
• To improve the knowledge about Lyme disease and the strategies for preventing tick-borne disease on the population level.
• To compare the rate of infection in the different sites nd the contribution of the proper knowledge to get an understanding of the risks of Lyme disease.

1.3 Research Question

1. What is the prevalence of Borrelia spp. infection in the two sites and what could be attributed to the difference encountered?
2. What factors have an impact on the distribution and the density of Borrelia spp.?

2.0 Materials and Methods

2.1 Research Philosophy

This research adopts the positivism research philosophy, which assumes that we can arrive at the truth by observing and measuring the phenomenon in an orderly manner. Real-time PCR is used for the purpose of determining Borrelia spp. prevalence in I. ricinus ticks, and this approach largely reduces bias. The study adopts a positivist approach and uses methods that are already defined in scientific research to identify Borrelia spp. and statistical methods to test for association (Hansford et l. 2022). Due to the data about infection rates, conducting hypothesis testing as a reference to the findings of the study follows positivism that is focused on the objective results and data. Thus, the objective is to produce practical, definitive conclusions about the prevalence of tick-borne pathogens according to the two sites that were chosen.

2.2 Research Approach

The research method used in the study is a deductive research method in which the researcher tests a hypothesis that is guided by theories and knowledge. It started with the idea that there is a difference in the “prevalence of Borrelia spp. for the tick population in the area of the Lake District and in Kielder Forest”. The hypothesis is based on the results of real-time PCR and statistical analysis of infection rates with regard to the above factors to compare the findings and provide confirmation or rejection. It also makes sure that the analysis process is impartial since it employs molecular biology methods in testing the samples from the ticks for Borrelia DNA.

2.3 Research Design

This paper also uses survey instrumentation and cross-sectional research design with the aim of establishing the “rate of Borrelia spp. in Ixodes ricinus ticks sampled from two geographical locations in the United Kingdom”. The most important limitation of the study is that it is cross-sectional in nature; this implies that data was collected at one particular time point across the total study period. This study plan involves a non-random sample collection of rodents from two different ecosystems; subsequently, the Borrelia spp. DNA will be identified based on the real-time PCR.

2.4 Research Materials

Various materials were used for capturing the “ticks, isolation of DNA, and real-time PCR”. Trials were carried out on the collection of tick using the conventional field collection and larval ticks; they were preserved in Eppendorf tubes for preparation. DNA extraction was done using the method described by Guy and Stanek (1991) in order to extract pure DNA contents that will be used for PCR reaction. The species-specific real-time PCR was then conducted using the Sensi-mix, fluorescent conjugated probes, forward and reverse primers, and the sterile water for the detection of Borrelia spp. with the target 23S ribosomal RNA gene. Specifically, in the current study, a real-time PCR machine was used for results evaluation. The “Chi-squared test and Fisher’s exact test” were performed in order to compare the infection rates in the different sites. All about material used herewith tested affirmatively and conclusively well fitting the molecular diagnostic affirmation to be accurate, perfect and replicable.

2.5 Research Method

The objective of the study was to evaluate the abundance of “Borrelia burgdorferi in Ixodes ricinus ticks in two areas”, that is, Gummers How and Kielder forest. The study entailed capturing and analyzing the ticks using predefined procedures and laboratory analysis on infection rates at the two sites with a view of determining any differences. Both the locations were searched for the ticks using the standard drag sampling technique with a white cloth on which the ticks crawled in the search of the vegetation because they are known to hoist themselves on an object that resembles vegetation when they are in the group stage. The collected ticks were then stored and transported to the laboratory for identification. DNA was extracted from each tick and tested for “the presence of Borrelia burgdorferi s.l using PCR”. The test result was noted as positive or negative depending on whether the tick was positive or negative for the B burgdorferi s.l. To conduct an analysis of the data collected, statistical tests were employed. For the first hypothesis, one-way ANOVA was done in order to find out if there was any ‘significant difference in the mean prevalence’ of infection between the two sites. Besides, the “Chi-Square test and Fisher’s Exact test” were used to analyze the correlation of location and infection status. The statistical discriminant performed with Chi-squares applied a test of independence that indicated the relationship being examined to be not statistically significant. The Fisher test used for small sample statistics precisely calculated the measures of association. The graphical representation to determine the proportion of infected ticks was produced to reflect those of Gummers How study in addition to the confidence interval. The results obtained for infection rates did not differ significantly; thus the observed differences could have occurred due to chance.

2.5 Data Collection Method

There were two implementable steps in the context of data collection: field sampling and laboratory analysis. Ixodes ricinus nymphs were obtained from the areas of the Lake District, Cumbria, and Kielder Forest in the Borders. Females and males of these ticks were collected in Eppendorf tubes; later they were labeled and preserved in the respective manner before DNA extraction. Extraction was done according to the procedure laid down by Guy and Stanek in 1991. Detection of the Borrelia species was done specifically for this study using the real-time PCR assay employing a 23S ribosomal RNA gene. These results were noted as positive (P), negative (N) or, absence of any colour change as control (C). To justify the comparison of the infection prevalence of the different sites, Chi-squared tests were carried out in Excel, to assess the tests’ reliability. This kind of approach offered precise and factual data on the distribution of Borrelia spp. in various populations of the test ticks.

2.6 Research Ethics

This study complied with the ethic standards in scientific research. This eliminated any ethical issues of harming or using vertebrate animals/ fellow human beings and getting their consent. These procedures were implemented in taking the ticks to ensure that the environment was not interfered with in a negative way. All the tests were performed under appropriate biosafety level standards to avoid contamination of infectious materials. To ensure that the results obtained from the PCR assay were genuine, there was blinding of the samples to eliminate bias. There was no need for deriving ethical approval for using ticks for molecular analysis; however, the study maintained the basic ethical standards of being translucent and replicable. The results will be described factually to enhance the insight of public health practices concerning Lyme disease consequences, as well as adhere to other ethical principles of scientific research.

3.0 Results

3.1 Findings

ANOVA Experiment

The design used in this analysis was one-way Analysis of variance or ANOVA in order to compare the means of two groups; Gummers How and Kielder Forest. The significance of the study was to analyse the difference, if any, that exists between the two. As such, since the “P-value (0.11875) is greater than the standard alpha level of 0.05”, it is consequently impossible to reject the null hypothesis. Different values are obtained through the experiment, including values of F and P, respectively 2.464 and 0.11875. The value of F critical equals 3.9097 which is acquired through ANOVA experiment. They revealed that score results do not differ from the scores attained by the control group, indicating that there is no difference between the two groups. Similarly, the F-value, which is 2.464, is less than the F critical value of 3.9097, ‘meaning there is no significant difference between group variance’. The variance of Gummers How is 0.0777 while that of Kielder Forest is 0.0247; hence, the dispersion of data at Gummers How is wider than that of Kielder Forest. However, according to this ANOVA test, the difference in means of the two groups (0.0833 and 0.025) can be rejected, resulting in a lack of adequate statistical evidence to warrant the rejection of the null hypothesis (Olsthoorn et al. 2021). In general, this means that if any differences as such were noted, they could have been a result of randomness rather than because of the targeted differences highlighted here.

Chi Squared Experiment and Fishers Experiment

The Chi-Square test is the method used when checking the relationship between two nominal variables, Positive and Negative across Gummers How and Kielder Forest. The value of chi-square equals to 0.0941. A smaller Chi-Square value means that the distribution of the observed data is nearly equal to the distribution in the expected pattern, which was recorded for different groups. Since the Chi-Square test is used to check independence, this low value suggests that the location, Gummers How and Kielder Forest, and the outcomes, positive and negative, are not related. The following is the tabulation of the forecasted values and the actual observed values: positive values; 3 and 4 with 60 and 76 (Pawełczyk et al. 2021). These values are nearly similar, and this implies that any variance is marginal. With regards to positive and negative cases, they are approximately equal in both the Hill Manning and Morningside facilities, showcasing that the place of employment does not significantly influence the occurrence of positive cases. Therefore, the null hypothesis stands invalid since the value of Chi-Square is very low.

The formula is used to get the values of Fisher‘s test as the following

Here, the values of different variables are a=5, b = 55, c = 2, d = 78 and the value of n equals to 140 which is total sie of the sample. Whenever sample sizes are small, ‘Fisher‘s Exact Test’ is preferred because it gives probabilities exact of association of the variables made of categories. In this case, it was used to test whether or not the percentages of positive and negative cases differ between Gummers How and Kielder Forest. The value of P approaching 1 means that the location of test has no systematically different outcome (Zając et al. 2021). Therefore, it can also be concluded that whether a case is positive or not does not necessarily depend on the area. Further, since Fisher’s test is more appropriate for use with small sample sizes than the Chi-Square test this implies that can draw a more justified conclusion that the observed differences are statistically insignificant. Furthermore, the obtained results of Fisher’s test indicate that there is no significant relationship between the location and results as well, supporting the conclusions derived from the Chi-Square test. This goes on to imply that the percentage of positive results is not in any way corresponding with location.

3.2 Analysis of Findings

The figure has indicated the trend of ‘the distribution of Borrelia burgdorferi s.l. in Ixodes ricinus’ in the area of the Gummers How site. The y-axis, as for the other two graphs, shows the positive outcomes, marked as a percentage; the x-axis defines the site. The bar in the graph represents the prevalence of the ticks which tested positive for Borrelia burgdorferi s.l and the T represents variability or confidence intervals. Percentage of positive results on the premises was found to be 5%, while the error margin being of 25%. The fact that there is a large error bar, may indicate that the distribution is large, meaning that a small sample size is used, or that the true prevalence is highly uncertain (Gandy et al. 2024). Due to the statistical tests conducted earlier, including ANOVA, Chi-Square, and Fisher’s tests, it became clear that there is no statistical significant difference between sites with regard to the infection rates. This graph supports those conclusions as the low value of the estimated incidences with the large confidence intervals indicate that the infection might have been detected by mere chance rather than as a causal feature.

4.0 Discussion

From the current study, using ‘ANOVA, Chi-Square, and Fisher’s Exact Test’, there is no statistical difference in the occurrence of ‘Borrelia burgdorferi’ s.l. in both the observed sites, Gummers How and Kielder Forest. From the ANOVA table, the p-value of the result is 0.11875, indicating that it is greater than the alpha of 0.05; therefore, the ‘null hypothesis is not rejected’, which means that the mean difference between the two sites is not significant. Also, when it comes to the F-test, the F-value equals to 2.464, while the F-critical equals to 3.9097, which also indicates that the difference is not significant (Sands et al. 2022). The value obtained from the Chi-Square test for screening outcome distribution with a value of 0.0941 indicates that the observed distribution of positive and negative cases are close to the expected distribution, and hence there is no significant relationship between location and infection rate of COVID-19. Fisher’s Exact Test, especially when dealing with low numbers, also conducted a test to determine that the p-value was approximately 1, which, once again, supported the notion that infection rates are similar between sites. The bar graph assists these statistical results, showing that the prevalence is low and has a big standard error which means that there is high variation. This would indicate that any differences that are picked may not actually be as a result of site differences, but rather they are just due to random chance of occurrence of Borrelia burgdorferi.

Conclusion

This study was designed with the objective of identifying whether there was a statistically significant difference of ‘Borrelia burgdorferi within the species of Ixodes ricinus’ between the two places under consideration; Gummers How and Kielder Forest. The statistical results such as ‘ANOVA, Chi Square, and Fisher's exact test’ more or less showed no distinction between the two sites. Through the analysis of variance (p = 0.11875) as well as through Fisher’s test, the results are near to 1, which indicates that almost all fluctuations may be attributed to chance. One has to add that the error bars in the prevalence graph are rather wide, which means that the very estimates of the infection rates are rather uncertain. In summary, this study finds out that the location does not have any effect on Borrelia burgdorferi in Ixodes ricinus, though a larger sample size must be used in further research for accurate results.

Reference List

Journals

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