Chapter 2: Materials and Methods
X- ray film development
After the series of washing steps illustrated above, 5ml of solutions ECL1 and ECL2
(Amersham, UK) were spread sequentially over the membrane, which was then incubated at room temperature for a minimum of 1 minute. The membrane was then placed in between two stretched layers of a clean, transparent cling film, secured within an exposure cassette and taken into the dark room where the development of the X-ray film would take place, enabling the reading of the results of the experiment. Once in the dark room, the X-ray hyperfilm (Amersham, UK) was ‘exposed’ (by being put in close contact) to the membrane (secured within the cling film layers) for 7 minutes, within the exposure cassette, for detection of any possible chemiluminescence signal originating as a reaction between streptavidine-conjugated horseradish peroxidase and ECL, wherever a sample’s DNA
hybridized with a complementary oligonucleotide sequence. At that point, the X-ray film was removed from the cassette and developed using an (Ecomax X-ray film processor, Germany). An example of the results obtained from the screening of a set of samples collected from cattle at KGR is shown in Figure 2:5 below. Once each hyperfilm X-ray was developed, results would be read by juxtaposing the film on a grid printed on an A4 sheet, designed using Adobe Photoshop CS 5.1, specifically for the membranes used in this study.
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oriented perpendicularly compared to the disposition of the catch-all and species-specific probes (therefore, ‘vertically’, compared to the ‘horizontal’ sense of orientation of the probes in Figure 2:4). For each sample, results were read by examining the presence of squared spots. In particular, the presence of squared spots, in correspondence either with catch-all or species-specific probes, indicates a positive result, regardless of the intensity (i.e. several shades of black-grey). In fact, in presence of a uniform concentration of oligonucleotide probes (which in the case of the membranes here used were of 400 μM), the intensity of a positive signal depends on the concentration of the DNA of the respective target
microorganism within the tested sample.
Importantly, in order to be considered as reliable, all species-specific positive signals need to be accompanied by a spot in correspondence of the ‘catch-all probe’ pertaining to their respective genus (e.g. Theileria mutans-specific signals need to be accompanied by ‘Babesia/Theileria catch-all’ and ‘Theileria catch-all signals’). Results characterized by a signal at the catch-all probe without a signal at any species-specific probes of the same genus are indicative of the detection of new species or of a different or novel strain compared to those for which the oligonucleotide probes were fixed on the membranes. Samples yielding this type of results (i.e. ‘catch all’-only positive for any of the tested genus of tick-borne microorganisms, not accompanied by a species-specific positivity) were therefore subjected to DNA purification and sequencing. At the same time, no spot of any intensity of black-grey should be detected within the vertical lanes corresponding to the negative and wash controls. Contrarily, the presence of signals within these lanes is indicative of contamination. Results characterized by species-specific signals (of variable intensity), not accompanied by a catch- all signal for the respective genus, are to be considered as ‘doubtful’ and warrant a replication of the RLB protocol as described above.
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Figure 2:5: RLB results after X-ray development of hyperfilm for cattle samples 1-35
[E/A = Ehrlichia/Anaplasma positive control (i.e Ehrlichia ruminantium); T/B = Theileria/
Babesia positive control (i.e Theileria mutans). R = Rickettsia positive control (i.e Rickettsia conorii). B = Bartonella positive control (i.e Bartonella hensele). - VE = (MilliQ water
control) and W0, W1, and W2 are blank white paper negative control]
Membrane stripping for re-use
Following the development, the ‘stripping’ of the membrane was carried out at the end of the RLB hybridization protocol, to remove DNA attached to the probes, enabling the re-use of the blotting membrane. Before starting, the temperature of the shaker incubator was set on 70C with at least 500 ml of 1% SDS for not less than 30 minutes. The membrane to be stripped was then put in a plastic tray, adding approximately 100 ml for the 1% SDS solution pre-heated at 70C, and incubating for 30 minutes in the shaker incubator at 70C under energetic shaking. Following these 30 minutes, the 1% SDS solution was discarded and the same washing procedure was repeated for other 30 minutes, using the same conditions
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Following the removal of the second volume of 1% SDS, the membrane was then incubated with 80 ml of 20 mM Ethylenediaminetetraacetic acid (EDTA) solution, for 15 minutes, at room temperature under gentle shaking. The membrane was then placed in a storage plastic seal bag, containing approximately 50 ml of 20 mM EDTA, facilitating its preservation, and stored in a fridge at 4 C until further use. The plastic seal bag was always signed after usage to identify the number of times each membrane was used. On average, each membrane can be used up to 20 times (Gubbels et al., 1999).
PCR product purification
PCR products were purified using an Isolate II PCR and gel kit according to the
manufacturer's instructions (Qiagen). Four volumes of binding buffer were mixed with one volume of PCR product (typically 20 μl) then the mixture was added into the spin column. The spin column was centrifuged at 10000 x g for 1 minute and the flow-through was
discarded. The DNA that bound to the column, was washed by the addition of 650 μl of wash buffer followed by centrifugation at 10000 x g for 1 minute. Again, the flow through was discarded and any residual wash buffer was removed by centrifugation at 10000 x g for 2 min. After that, the DNA was eluted from the column into a 1.7 ml Eppendorf tube by the addition of 15 μl of sterile distilled water into the centre of the column, incubated at room temperature for 1 min, then centrifuged at 10000 x g for 2 min. Purified PCR products were stored at -20C.
Sequencing and phylogenetic analysis
Sequencing was performed commercially (Macrogen, Netherlands). Both strands of each PCR product were sequenced using the same primers as for a single round PCR while for a nested PCR the second round primers were for their amplification. The chromatograms (.ab1files) were visualised using ChromasPro software (version 2.1.4). Sequences were verified and, if necessary edited, and primer sequences were removed. Verified sequences were exported into the Molecular Evolutionary Genetic Analysis (MEGA) version 7.0 programme suite (Kumar, Stecher, & Tamura, 2016).
In addition to sequences generated in this study, other relevant data were imported into MEGA7 from GenBank to generate personal databases of relevant sequence data. Sequences drawn from these databases were assembled into multiple sequence alignments using
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alignments using the neighbour-joining method (Saitou & Nei, 1987), and visualised in the form of dendrograms using TreeView. The stability of branching orders proposed in these dendrograms was assessed using bootstrapping (1000 replicates) (Felsenstein, 1985).
Epidemiological statistical analysis Univariant analysis
Field data and results generated in the laboratory were entered and managed in Microsoft- excel. SPSS version 20.0 software program was initially employed for the data analysis. The overall prevalence of tick and tick-borne haemoparasites was determined by dividing the number of positive animals by total sample size and was expressed as a percentage. Similarly, the exact binomial 95% confidence interval (CI) were determined. Chi-square (χ2) test was used to assess the association of tick infestation between different variables and to test the null hypothesis for significant difference between age classes (i.e. young and adults) and sex classes (male and female) with regards to overall and individual haemoparasites infection. Effects were reported as statistically significant in all cases if the value is less than 5% (p>0.05).
Multivariant analysis
To investigate those factors that influence an individual’s probability of testing positive for infection with tick-borne haemoparasites, generalized linear mixed models (GLMMs) were used that assumed a binomial error term and a logit link. In all of these models, the owner of the cattle was used as a random effect to account for potential non-independence due to different owners employing different methods of control. Individual level-factors considered included coinfection with other haemoparasites, age of the individual (categorised as 0-24 months as young, while 24 months and above as adults), gender, haematological parameters including packed cell volume (PCV).
All analyses were carried out using R 3.4 software (R. Development Core Team, 2016) using either the glmer function from the lme4 package. Model selection was based on a backward stepwise model selection with variables dropped according to P-value, with only those variables significant at the p<0.05 level being retained in the final model.
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