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Ticks harbour several bacterial, protozoal, and viral pathogens that can cause severe infections in domestic animals and humans. Active observation of the tick vector can present insight into the occurrence and distribution of significant pathogens in the environment. Nucleic-acid based detection of tick-borne bacterial, protozoan, and viral pathogens requires the extraction of DNA (total nucleic acids) from ticks. Traditional methods for nucleic acid detection are restricted to extraction of DNA from a sample of ticks. By using Qiagen robot based protocol, DNA was extracted from (n=75) ticks and this demonstrate the presence of Borrelia afzelii and Rickettsia in individual, infected Ixodes ricinus ticks.
The study aimed at detecting the presence of Borrelia and riskettsia in
UK ticks using real time polymerase chain reaction (PCR) with primer and probe targeting cirtrate synthase (CS) and flagellinBb. Of the entire 75 ticks DNA samples tested using real time PCR assay, no positive result were detected.
The none detection of positive samples indicated the absence of Borrelia and Rickettsia in the ticks used for this research work and this may be due to the blood meal inhibition.
Ticks are bloodsucking, ectoparasites feeding from most terrestrial vertebrates. Ticks belong to the order Acarina, which contain mites and fleas. Spiders are more closely-related to ticks than other insects. Ticks transmit a variety of contagious agents that are pathogenic to livestock and can cause illness in human, and can also harm their host directly by inducing toxicosis. (Beugnet, 2009).
1.1 ENVIROMENTAL FACTORS AND EPIDERMILOGY
Two of classes of ticks parasitized livestock, the Argasidae (soft ticks) and the Ixodidae (Hard ticks). Even though they both share basic properties, argasids and ixodids vary in various ecologic, structural, reproductive, behavioural, feeding and physiologic patterns. (Bruce and John 2002). Certain tick's species particularly Ixodes ricinus parasitize livestock may survive numerous months and years, without feeding if the ecological conditions permits and This study focused on the (I. ricinus) tick and the detection of Borrelia, the cause of Lyme borreliosis and Rickettsia, the cause of various spotted fevers. (Pietzsch et al 2006)
Argasidae ticks are found in sheltering environment in the host, wood, caves and rocks and some argasid species are capable of surviving unfed for several years. (Evan 2002) these leathery parasites inhibit warm temperature or tropical environment with long dry seasons. Host are those that returned from time to time to either rest or breed there. (Hendson, M. 2000)
Ixodes ricinus are hard ticks (family ixodidae) due to the facts that gnathosoma comprises of sensory appendages, cutting, piercing andtearing appendages and hypostome that are clear and basically visible in the life cycle, the upper body surface are completely covered (male) by hardened scutum and the anal grooves arching in front of the anus are only seen in the genus ixodes. (Stafford 2004)
Ixodes ricinus ticks posses sensory organ, Hallar's organ situated on the front tarsi, used in detecting ecological stimuli such as carbon dioxide levels, humidity and changes in temperature, these was observed by questing. They also possess the exterior respiratory openings which are situated on a pair of spiracular plate. Hillyard, P.D., (1996)
1.2 LIFE CYCLE OF IXODES RICIUNS TICKS.
Ticks are broadly distributed in a variety of habitat including grassland, rough pasture, heath and deciduous woods. The absence or presences of ticks are extremely dependant on local conditions which are basically determined by humidity and sward height. The diverse range of vertebrate host which ticks feed affect about 240 species of natural domesticated mammals, reptiles and bird's species. (John 2006).
Ixodes ricinus, the sheep tick, are the commonest ticks in northern and central Europe. Ticks are typically a three host species and their complete development occurs approximately 3 years, passing through an eggs larva, nymph and adult stage(fig.1), taking one year, however the life cycle can differ in length between 2 and 6 years (Gray, 2009.). Blood feeding occur once in every stage and for a phase varies according to stage. Digestion of the blood meal and development to the next stage occurs while hidden deep in the vegetation. The larvae hatch from an egg batch of about 2000 and after a few days are ready to feed. (Wodecka, 2003)
Ixodes ricinus are hard ticks, as a result of the presence of the tough outer scutum, a hard sclerotized dorsal coat, this feature provides sexual dimorphism in the ticks as the scutum completely covers the idiosoma of the adult male, whereas the female incompletely covered revealing the alloscutum which allow the female ticks to engorge during blood meal. They are major vector for transmissible pathogens, transmitting above any other arthropods vector group. Disease transmitted by ticks is of huge medical and veterinary importance, (Table1). Ixodes ricinus in the UK is also the primary vector for Anaplsama phagocytophilum causing human granulocytic ehrichiosis and equine ehrichiosi. (Jongejan, 2004).
Table1. Ticks borne disease present in UK.
Rabbits, humans, rodents, dogs
Bovine and Ovine
Anaplasma Phagacytophilum complex
Wild ruminants, goats, sheep cattle
Babesia divergens, B.micrti
Humans and cattle
Louping ill virus
Red deer, sheep, hares, rabbits, humans, grouse
Recently, there has been a unusual increase in the prevalence of many ticks borne diseases, consequence, ticks are of paramount importance worldwide,
Because of the implications on both livestock and humans health.
The following is a simplified description of the life cycle of the tick, Ixodes ricinus. Figure1
Eggs are laid in early spring, hatch as larva through the spring/ summer, larva utilize birds and mice as their initial host, drop and develop into nymphs and undeveloped for six months, the undeveloped nymph stirs in spring and locate a secondary host in other mammals, humans and deer. After feeding on the blood meal the nymph drop off the host and fully grown into adult stage. The adult locates their tertiary host: dogs, deer's, sheep and humans, and then an adult enlarged out of all proportional to its original size after feeding. Fully developed once mate and finally drop off the host and finally the adult females lay eggs at two years old or beyond. (Gray 2003).
1.3 LYME DISEASE. (LD)
Lyme disease was first described in 1975 in three Connecticut town when 39 children and adults developed recurrent asymmetric pain in a few large joint and swelling, most often the knee. (Snydman 2011). Today, LD is the most common arthropods borne diseases in the United States, affecting over 170,000 people and has also been reported in Asia, (Ai, C.X et al 1998)Northern Hemisphere, (Masuzawa,2004) Canada, and Europe (Matuschka, F.R., 2004), Affecting humans including neurologic symptoms, facial nerve paralysis, flu-like symptoms (Fritz, 2003)
Borrelia burgdorferi, are the agent responsible for causing LB in animals and humans, is transmitted by the bites of infected Ixodes species ticks. Ticks removed from animals are tested in the laboratory. The intention of this study was to confirm the detection of the presence of Rickettsia or Borrelia in the British ticks. Testing of ticks for Borrelia is often requested of analytical laboratories in LD endemic area. Confirmation of B. burgdorferi positive ticks can speed diagnosis of the disease, if clinical sign develop, treatment can be initiated speedily, The disease is usually easy to treat, but if allowed to develop, several joint problems and neurological will result and can continue for years and there are several method used to detect Borrelia in ticks which consist of dark-field microscopy, traditional polymerase chain reaction (PCR) and real time PCR (Gray, 2002)
The geographic distribution or LD area governed by the ticks and its associated animals host forestry and farmers are particularly at risk. In UK the LD occurs mainly in the area that support large domesticated animals on which Ixodes ticks feeds. (O'connell 2005)
Ixodes ticks are vectors are common in woodland; moorland and heath also can live in semi-rural area bordering large inhabitants. They take a blood meal at each stage of their life cycle, attaching to their host by barbed mouthparts. An infected tick may transmit Borrelia towards the end of its feed, regurgitating infected saliva into the animal's skins. Reservoir hosts for the spirochetes include medium sizes mammals. (E.g. hares, field mice) birds, pheasants and gulls. Humans are minor host for ticks, and an infection occurs in early summer, late springs and autumn. The annual incident of LD can vary, depending on climate factors affecting ticks activity, population and density. (Stanek, G., et al 2003)
Nymphal are the major cause of humans Borrelial infection because humans can be an incidental host for the tick at any stage and even after a feed, they stay behind, and can be ignored by their host, ticks bites can be recognized since they do not cause significant irritation or pain. Those exposure to ticks can minimize their risks of infection by wearing protective clothing and taking into consideration use of insects repellent. Borrelia is possible to occur when ticks attached for less than 24-36 hours, prompt removal is an additional preventive measure. (O'connell 2005)
Borrelia can spread through bloodstream and lymphatics too many tissues and might migrate through the blood-brain barrier, evading the host's immune responses and autoimmune factors have been implicated in the pathogenesis of neuroborreliosis also to direct spirochaetal invasion and cytokine mediation and also antibodies produced in response to spirochaetal antigens may cross react with axonal tissue component. (O'connell 2005)
Three genospecies of B.burgdorferi senus lato are established as pathogenic, and Borrelia heterogeneity are significant in organotropism and disease presentation and merely B. burgdorferi sensu stricto emerge to cause humans infection in North America, other two that are pathogenic genospecies are found in Europe B.afzelii associated with later skin manifestation and B. garinii associated with neurological complications. (O'connell 2005)
1.4 ROCKY MOUNTAIN SPOTTED FEVER (RICKETTSIA)
Rickettsai rickettsii is an obligated intercellular bacterium. In the series of elegant experiment, Burgdorferi (1980) show that wood ticks fed on infected guinea pigs could transmit RMSF to noninfescted guinea pigs during subsequent feeding and also demonstrated that ticks (R. rickettsii) circulates in a zoonotic cycle that transstadial and transovarial maintenance by its arthropods hosts, Ixodid ticks and horizontal transmission to its vertebrate host, which include a wide range of medium and small size mammals. Other rickttesia species (Table 2) known to be pathogenic for human. ( Burgdorferi 1980)
Rickettsiae (tick typhus) are obligate intracellular gram-negative bacteria, which lining in blood vessels, the onset of symptoms is sudden with fever, chills, muscle pain, headache, high and then a rash; blood vessel inflammation are common. The members which have a worldwide distribution, are mainly associated with tick, fleas and mites, and the distribution of the rickettsioses will be identical to that of its competent arthropod vector when the tick serves as a reservoir. (Parola, 2005)
The genus riskettsia species comprise the cause and often fatal humans disease, including the etiologic agents of (RMSF) Riskettsia riskettsii, Risketttsialpox (Riskettsia akari) Table 2 also still shows other riskettsia species appear to cause little pathogenesis in vertebrates and apparently are maintained in nature are almost exclusively by transstadial and transovarial transmission in the ticks host. These Gram-negative obligate intracellular bacteria are all morphological similar within the cytoplasm of host cell. Riskettesia species are grouped into one or two serogroups typhus(TG) and spotted fever(SFG). (Bruce 2002)
Table 2 RICKTTESIAE DISEASE IN LIVESTOCK AND HUMANS
Primary vertebrate hosts
Medium and small sized mammals and rodents
North, South, and central America
Oriental spotted fever
Other Medium and small sized mammals and Rodents
Queensland tick typhus
Field vole and house mice
Korea, USA and other area
Rodents, cats, humans and other small sized mammals
Europe, India, Middle east and Africa
AIM AND RATIONALIZATION
This project aimed to indentify the presence of Rickettsia and Borrlia in ticks found in Richmond Park and among ticks removed from wildlife species in the United Kingdom. It was anticipated that this study would indentify area of infection activity and consequently indicate the risk of exposure to ticks and their transmitted disease.
Richmond Park It popular with tourists' attraction hundreds of individual visitors a month. The park is a home to a huge number of deer which act as primary host for the I. ricinus. Its exceedingly important to conducts these study over the summer as this is the peak time of social activities within the park, the assumption that weather conditions is nicer and however more people will visit the park and this also correspond with the life cycles of the ticks resulting in peak larval activity for the period of the summer season.
2.0 METHOD AND MATERIAL
2.1. MEADOW RESEARCH
The research sites were situated in london a national nature reserve (Richmond park) containing 955ha of primary oak tree (Quercus robur), acidic grassland at the heart of the park and woodland. Richmond Park is a site of special scientific Interest (SSSI) due to its population of dead wood fauna, which are the Red data species, 135 beetles are recorded in the park according to (B.P.J., Greenfield 2011).
In a lately published survey (Jones and Reeve, 1996, p.81-90) the authors discovered that, Richmond park were habitat for five species of significant host for I. ricinus and reservoirs for B. burgdoriferi, pygmy(S. minutus), common shrew (Sorex araneus), bank vole (Myodes glareolus), wood mouse (Apodemus sylvaticus) and field vole (Microtus agrestis).
2.2 TICKS COLLECTION AND PREPARATION
Ticks were collected from Richmond Park by flagging vegetation with blanket, these were washed with 70% ethanol and sterile water, suspended in 200 sterile phosphate buffer saline (PBS), homogenized by loading the ticks (n=75)in Bio sample bench top homogenizer at the speed of 5.0m/60second and the homogenized ticks sample were stored at 3-4 degree Celsius for the next stage of analysis.
DEOXYRIBONUCLEIC ACID (DNA) EXTRACTION FROM TICKS
DNA extraction was carried out with Qiacube and DNeasy blood/tissue extraction kit, DNA were extracted from n=75 ticks samples using the automated robot Qiacube (Qiagen), kits (fig 2) according to the manufacturer instructions. The method was automated on the qiacube DNA extraction robot (Qiagen Roche) following the homogenizing step and. manual collection of the supernatant
Diagram of DNA tissue procedure
FIG.2 PURE GENOMIC DNA
The extracted ticks DNA was stored at -20 °C until further analysis by PCR amplification.
REAL TIME POLYMERASE CHAIN REACTION. RT-PCR
Polymerase chain reaction (PCR) is a method that allows exponential amplification of short DNA sequence within a longer double stranded DNA molecules, PCR entails the use of a pair of primers , each about 20 nucleotides in length, that are complementary to a defined sequence on each of the two strands of DNA. (Margaret, 2010)
PCR were intvented, as a fairly standard procedure to amplify the DNA template, which can amplified a small amount of template DNA into a larger quantities in a few hours. (Purves, 2001) Recently a new method of PCR quantification was invented, and were called real time polymerase chain reaction because it allows the scientists to actually view the increase in the amount of DNA as it amplified, RT-PCR are of different type SYBR Green , molecular beacon and TaqMan real time PCR. (Purves, 2001)
RT-PCR is based on the detection of a fluorescent generated proportionally during the amplification. The key to the detection system is the chemistry, probe (TaqMan) are created to anneal to the target sequence linking the traditional forward and reverse primers, the probe was attached at the 5' end with a reporter fluorochrome (6-FAM) and a quencher fluorochrome added at the 3'end. The probe was designed to contain a higher Tm than the primers and during the extension, the probe must be 100% hybridized for the success of the assay. However as Taq polymerase extend the primers, the intrinsic 5' to 3' nuclease action of Taq degrades the probe, releasing the reporter fluorochrome. And total of fluorescence released during the amplification cycle is proportional to the total of products generated in each cycle. (Persson, 2005)
The sensitivity of detection permit acquisition of data when PCR amplification is still in the exponential phase it is determined by the identifying the threshold cycle (Ct)at which the reporter dye discharge concentration rise above the background noise. The sensitivity exists because quantitation of the gene sequence is determined by the Ct, which was calculated during the exponential phase of the reaction. High specificity is conferred by the obligation of three oligos to anneal to the DNA before data are collected. Benefit of providing the quantitative RT-PCR technology in a core facility setting in reduction of labour cost and time. (Raeymaekers, 2000)
The TaqMan DNA Polymerase reagent kit (invitrogen) has been satisfied for our use in this project and it allows flexibility in preparing the different master mixes that are needed for the variety of primers and probe used. Real time monitoring of the release of fluorescence, several times during each cycle allows collection of abundant data. After 40-50 cycles, data are processed by the software within seconds. RT-PCR provides an accurate and a rapid method for determining level of specific DNA and RNA sequence in various tick samples. (Rickert, 2004)
REAL TIME PCR GENE SPECIFIC
The real time polymerase chain reaction was performed in a Stratagene thermocycler, the real time PCR reaction were mixed before and after PCR was handle in a safety cabinet which was ultraviolet (UV) illuminated before and after use, to eliminate contamination. PCR master mix was prepared to give a final volume of 25µl comprised of 1- buffer (Invitrogen), 1000nM of both rrs primers (Sigma Genosys) 2ul of DNA extract, negative control (sterile water )and positive control were included in the experiment. The sample was tested for the presence of Borrelia and Riskettsia Spp., DNA by PCR using the primers specific for each of the pathogens. (Gelmini 1997)
Riskettsia were detected by amplifying a fragment of the citrate synthase primers of Risketttsia spp., according to Nilsson et al. (1999) with the forward primer 5uM CS F (5'-TCG CAA ATG TTC ACG GTA CTT T-3') and the reverse primers CS R(5'- CAC AAT GGA AAG AAA TGC ACG A-3') with 100nM probe (Sigma Genosys) CS P FAM-TGCAATAGCAAGAACCGTAGGCTGGATG-BHQ1. 0.2mM dNTP's (Invitrogen), Taq DNA polymerase (Invitrogen) at 5units/µl, ROX (Stratagene) 1mM(1:500) 0.375µl, 7.475µl of water and 2µl of DNA template. The initial denaturising took place at 95°C for 10 minutes, following by the 50 cycles of amplification at 60°c for 60 minutes, together with the denaturising at 95°C for 15 seconds and annealing temperature for 45 second at 60°C.
PCR BORRELIA DETECTION
PCR master mix was prepared to give a final volume of 25µl comprised of 1- buffer (Invitrogen), 5mM MgCL2 (Invitrogen) and 700nM of both rrs primers (Sigma Genosys), negative control (sterile water )and positive control were included in the experiment. The detection of Borrelia spp in ticks DNA was performed using the RT-PCR primers targeting the flagellin gene (flaBb). The forward flagellin primers for Borrelia were flaBb (5'-GGG AAG CAG ATT TGT TTG ACA-3') and the reverse flagellin primers were (5'-ATA GAG CAA CTT ACA GAC GAA ATT AAT AGA-3') with 100mM flagellin probe(Sigma Genosy) P FAM-ATGTGCATTTGGTTATATTGATCTTGATCATCAA-BHQ1. 0.2mM dNTP's (Invitrogen), Taq DNA polymerase (Invitrogen) at 5units/µl, ROX (Stratagene) 1mM(1:500) 0.375µl, 7.475µl of water and 2µl of DNA template
The initial denaturising took place at 95°C for 10 minutes, following by 40 cycles of amplification at 58°C for 30 seconds, Together with the denaturising at 95°C for 15 seconds with the annealing temperature for 45 seconds at 55°C.
RT-PCR MASTERMIX WITH Taq
CONFIRMATORY TEST FOR BORRELIA
The extracted ticks DNA were diluted in 1:10, 1:100 and 1:200 with distilled water in three fold dilutions by using phosphate buffer saline (PBS) sample as spike with the homogenized tick tissue. Samples were amplified and analysed on the real time polymerase chain reaction, using the procedure for Borrelia amplification with tick DNA by PCR.
The real time polymerase chain reaction amplification was used to determine the presence of Riskettsia and Borrelia species in ticks DNA extraction. The findings were negative for both pathogens. Furthermore a confirmatory test were perform for the Borrelia species using threefold dilution but the findings still remain negative with the ticks used in this research work. (Massung, 2003)
PCR LABORATORY ANALYSIS STATISTICS
The RT-PCR amplification plots explained that, a positive sample were expressed as comprising a cycle threshold (Ct) value between 4 and 29 cycles. Negative samples were translated as having a Ct value less than 1 no amplified rise as the in fig.4. Establish on this threshold, among 75 sample of DNA extracted tick samples, analyse no positive reaction for PCR detected , 15 samples of PCR negative were examine in threefold serial dilutions and the result remain the same as negative
Fig.4 shows the Amplifier plot for Rickettsia (CS).
The utility of the Real Time PCR TaqMan assay form the rapid screening of field collected ticks for Borrelia and Rickettsia was assessed by analysing 75 sample DNA ticks. DNA extract from each ticks were analyzed by using RT-PCR assay (flaBb and CS) and TaqMan. With all (n=75) none were positive. DNA purified from Borrelia was spike into reaction mixture containing no action template DNA. DNA extracted from ticks that tested PCR negative for Borrelia. Threefold serial dilution for samples were analyzed in triplicate with real time PCR
The analysis was done as a confirmatory analysis for Borrelia but the result were still negative, the amplification plot was not shown
The study were aimed to indentify the presence of Rickettsia or Borrelia In UK ticks, using real-time PCR to detect them The result of the real- time PCR showed a Rickettsia and Borrelia significant infection rate of 0% among 75 samples of UK ticks used. The addition of positive control in this assay guarantees that negative PCR product representing a Rickettsia or Borrelia will be negative samples. The reason of the negative result in RT-PCR amplifier might be from the tick's blood inhibition and poor quality of real Time PCR, Template, primers and its effectiveness. According to (Eremeeva, Dasch and Silverman) these authors' in their recent work suggested that the traditional PCR techniques lack the sensitivity to identify infection once there are low statistics of the pathogens in blood samples. The standard dilution Series access transfer a good estimated of the efficiency of the PCR analyze. Ticks DNA sample may contain inhibitory substances that are not present in standard base on the purifications template this may reduce the PCR efficiency and resulting to a negative to the samples amplifier. Dilution of the samples (ticks heme) will tends to reduced the inhibition and estimates the PCR efficiency. Common PCR inhibitors are heme (blood), lipids, IgG, collagen (tissue) (Al-soud 2000; Izreali 1991:Akane1994)
When the inhibitor are hard to remove by dilution method, its better to employ a standard additional dilution (stahlberg2003)
Van 2005 and Rutledge 2004 suggested that estimating real-time PCR response efficiency because it will allow more determination in an extension in a number of targets in samples
The Mowry 1999 and Mhlanga 2001 suggest that using different kit to assay for detection of Borrelia in ticks like cultivation in BSK medium, dark-field microscopy, an immunofluorescence assay, and PCR that a comparison of the mean infection rates for studies in which at least one of these methods was used for detection of Borrelia in ticks revealed no significant difference in either nymphs or adults and the highest infection rates were obtained from place with PCR in Bulgaria, Croatia, southern Germany, Latvia, and Slovakia. In an analysis
The rates of infection of ticks with Borrelia were correlated with the latitude or longitude of the sampling site in every study. For this purpose, the coordinates of the sites of tick collection were determined. In studies with a large collection area, the means of latitude and longitude were calculated. Coordinates were transformed to decimal values. Negative longitudes represent the zone west of Greenwich, England, and positive longitudes represent the zone east of Greenwich say s the author
metaanalysis provides a versatile alternative to the more traditional review methods and allows quantitative conclusions to be drawn. The prevalence of Borrelia disease in ticks is one of the most essential components of risk assessment for LD. In recent years, many studies of the rate of infection of ticks with Borrelia have been accounted. (Mhlanga 2002) Analysis of data from 155 records of studies conducted in Europe showed that the overall mean infection rate was 13.6% and that the rate of infection of adult ticks was significantly higher than that of nymphs, which was observed in the majority of the studies and is explained by the fact that host-seeking adult ticks had had two blood meals on different hosts. These data are in line with the data reviewed by other workers (Hubalek, 1998 and Gray 1998)
Recently, a B. miyamotoi-like Borrelia species was detected in I. ricinus ticks in Europe (99 Fraenkel2002). Therefore, the rate of infection of ticks with spirochetes belonging to the B. burgdorferi sensu lato complex may be overestimated by some detection methods( jensen, 2ooo)
I would like to thank the Almighty God for the strength and protection in the cause this project work. I also offer my sincere thank to my supervisor Dr. Sally J. Cutler (University of east London) for her ongoing support, help in the field collection of ticks, encouragement and assistant she has provided me with throughout the entirety of this project; I would also like to extend my thanks to Nigel Reeve (Richmond Park) for granting us permissions for the collection of ticks to conduct this project. Thanks to Ifeyinwa Maduka and Essen David-mack for their enthusiasm and their advice in cause of this work. Finally, appreciation to my dearest brother Sir Sunny Igboanuzue, for his honourable and financial support.