Study area and study design
Eight locations in two separate counties were investigated for p. multocida infections, five locations in Marsabit County and three locations in Turkana County. Marsabit County (37°58' E, 2°19' N) is situated in northern Kenya bordering Wajir County to the east, Ethiopia to the north, Isiolo County to the south east. Its size is 70,961km2 and a population of about 459,785. Marsabit camel population is approximately 132,215. Turkana County (3° 09′N 35 ° 21′E) is located in northern Kenya bordering Samburu to the South-east, Marsabit to the East, Baringo and West Pokot to the South-west, Uganda to the west, and South Sudan to the north and Ethiopia to the north-east. It covers 68,680 km2 with a population of about 926,976. Turkana camel population is approximately 456,826 . The study was a retrospective study that used samples collected for diagnostic purpose.
A total of 132 samples (n=132) were collected, 61 EDTA blood in November 2018 at five locations viz Laisamis-moile, Nairibu, Malabot, El burumagado and Galas in Marsabit County, 41 EDTA blood and 30 nasal swabs in April 2019 at three location viz Nadapal, Lokolia and Lokore in Turkana County from randomly selected clinically sick camels population that had been affected by pasteurellosis like disease. A P. multocida Kabete isolate was used as a positive control and nuclease free water as a negative control. The authority to use the EDTA whole blood and nasal swabs archived at Central Veterinary Laboratories Kabete was sought from State Department of Livestock, Directorate of Veterinary Services in Kenya (Figure 8).
The frozen whole blood samples and swabs where retrieved from freezer and left to thaw at room temperature. The genomic DNA was extracted using Invitrogen PureLink Genomic DNA mini Kit Cat. No.k1820-01 for purification of genomic DNA by thermofisher Scientific. Water bath was pre-set at 55°C. To a sterile Microcentrifuge tube 200µL of frozen whole blood and nasal swab suspension was added then 20 µL of each proteinase K and RNase A were added, mixed by briefly vortexing and incubated for 2 minutes at room temperature. Homogenous solution was made by vortexing after adding 200µL of lysis/ binding buffer. The homogenous solution was incubated at 55°C for 10 minutes to promote protein digestion. DNA was precipitated using 96% ethanol and bound to a resin on a mini spin column, the column was centrifuged at 12000 rpm for 1 minute at room temperature. The flow through was discarded together with the collection tube. The spin column was placed into a new collection tube. The column was washed twice by adding 500 µL wash buffers 1 and 2 and centrifuged at room temperature at 12000 rpm for 1 minute and maximum speed for 3 minutes respectively to remove the lysis buffer. DNA was eluted using elution buffer . The DNA was kept at -20°C awaiting PCR analysis.
Detection of p. multocida by conventional PCR and multiplex PCR
Conventional PCR amplification targeting 460bp KMT1 gene fragment using specific primers previously designed by Townsend et al., (1998) (Table 5) was used to detect P. multocida. The component for DNA amplification master mix for P. multocida in a total volume of 18.0 µl were nuclease free water 12.2µl, 5x PCR buffer 2.5µl, 10mM dNTPs 0.5µl, 10µM forward primer 0.5µl, 10µM reverse primer 0.5µl, 3mM MgCl₂ 1.5µl and Taq DNA polymerase 3unit, 0.3µl. The amplification conditions were initial denaturation at 94°C for 3 min followed by 30 cycles of denaturation at 94°C for 45 sec, annealing at 55°C for 45 sec, extension at 72°C for 45 sec, final extension at 72oC for 6 min, holding at 10oC until removal. 5µl of PCR products was mixed with loading dye and analysed by gel electrophoresis on a 1.5% agarose gel. Multiplex PCR was applied for capsular type to differentiate P. multocida based on the genetic organization of the lipopolysaccharide .The capsular specific primers are designed for detection of capsular gene sequences, characterization and analysis of capsular groups . All P. multocida KMT1 gene confirmed positives were subjected to multiplex PCR using specific Primers previously designed by Townsend et al. (2001) for capsular group’s determination (Table 5). DNA amplification was carried out with a reaction volume of 20.0 µl containing nuclease free water 4.0µl, 5x PCR buffer 10.0µl, 10mM dNTPs 1.25 µl, 10µM primer pair each 1.25 µl, 3mM MgCl₂ 1.5 µl, Taq DNA polymerase 3unit 0.75µl. Amplification conditions were initial denaturation at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 1min, annealing at 55°C for 1min, extension at 72°C for 1min, final extension at 72°C for 7min, holding at 10°C until removal. 5µl of PCR products was mixed with loading dye and analysed by gel electrophoresis on a 1.5% agarose gel.
DNA sequencing of pasteurella multocida
Twenty one (21) PCR products of the isolates amplified using oligonucleotides targeting KMT1 and hyaD-hyaC, bcbD, dcbF, ecbJ, and fcbD capsular genes were sequenced, some at International Atomic Energy Agency (IAEA) Seibersdorf Laboratories, Vienna, Austria and others at inqaba biotec Africa’s genomic company, South Africa using Sanger dideoxy sequencing procedure .
Sequence alignment, blast analysis and phylogenetic analysis
BLASTn tool of the NCBI GenBank database was used to analyse the sequenced DNAs. The correct species identification was arrived at by comparing the query nucleotides sequences with GenBank database available sequences. The closest BLASTn match was used to confirm the species identification to the homologous sequences found in the GenBank database. The sequences were viewed using chromas version 2.6.6  and assembled using Geneious prime program version 2020.2.4  to allow for editing of the assembly and creation of consensus sequence. BioEdit software [15, 16] was used to import, align sequences, save aligned sequences and BLAST on NCBI (National Centre for Biotechnology Information) for sequence similarity. MEGA X  was used to construct phylogenetic tree by Maximum Likelihood method to determine relatedness. The internal branches statistical significance was inferred by bootstrapping with 1000 interactions.