Study Areas
Nairobi County
It is a highly urbanized county and the commercial and administrative capital of Kenya. It has an area of 696.3km2 with a high human population density of 4800/km2 (16). There is a high level of farming activities, especially in the peri-urban zones. This includes semi-nomadic pastoralism of cattle, sheep, and goats, intensive rearing of dairy cattle, and poultry (16). Some districts within the county have significant numbers of free-roaming populations of goats,sheep,chicken, and other poultry e.g. ducks. Nairobi National park, located within the county is home to a large and diverse wildlife population, Cape buffalo, Maasai giraffe, Grants zebra, African leopard, lion, eland, impala, cheetah, etc (17). Sections of the park borders are porous allowing constant interaction between wildlife, livestock (including dogs), and human beings (18). Anecdotal reports and published studies indicate that there is a large population of dogs, both stray and owned within Nairobi County. Some of the owned dogs are not kept under confinement and are allowed to roam freely. The dog population comprises both exotic and indigenous breeds with the majority being indigenous (6, 19).
Mombasa County
The county is situated along the Indian Ocean coast of Kenya and consists of Mombasa Island and the immediate surrounding mainland areas. It has an area of 294.9km2 of which 65km2 is water. It has a human population of about 1.2 million. It is a highly urbanized county and is the 2nd largest city in Kenya (16). Farming activities are carried out in the peri-urban zones and include intensive poultry (broilers & layers) and dairy farming, free-roaming cattle, sheep, goats, and chickens are also observed in the suburban areas (16). There is a large population of dogs both owned and stray, a large percentage of the owned dogs are allowed to freely roam around (20).Commercial/semi-commercial and subsistence fishing is carried out in the Indian Ocean waters around Mombasa. Mombasa also serves as a landing and processing zone for commercial fishing vessels (16, 21).
Nakuru County
The county is located in the Rift valley region. Naivasha town within Nakuru county has a human population of 181,966.Naivasha area is dominated by major geographical features such as Lake Naivasha, a freshwater lake, and Mt. Longonot with an elevation of 2,776m which is part of and surrounded by the Mt. Longonot National Park. The Park hosts a variety of wildlife including buffaloes, giraffes, plains zebra, Thomson's gazelle, and hartebeest (22).Naivasha is an important livestock farming area that includes intensive dairy cattle, poultry rearing and commercial beef cattle ranching. There is a significant population of free-roaming, and backyard cattle, sheep, goats, chicken and pigs. Nomadic pastoralism of cattle, sheep, and goats is also carried out within the area. There are significant fishing and related activities at L.Naivasha which is also an important wildlife area with hippopotamus and numerous bird species (16). There is a large population of dogs, both stray and owned especially in the peri-urban areas where a significant proportion of the owned animals are allowed to roam freely (23).
Sampling
The study employed a descriptive design, where sampling was opportunistic. A total of 143 dogs were sampled. In all 3 counties, samples were collected from dogs presented at the shelter facilities of the Kenya Society for the Protection & Care of Animals®.The samples were collected from both apparently healthy dogs with others being symptomatic with a variety of ailments.These dogs were of diverse backgrounds including stray, loosely owned, surrendered, and confiscation cases. The dogs were also of varied age, sex, and breed (24). Age assessment was carried out using a combination of criteria, including teeth, size of the dog, hair coat, the state of ocular lens (46, 47). Sex was determined by visual inspection of the external genitalia (48). The breed was established by visually comparing the dog's physical attributes against established breed characteristics (http://www.eastafricakennelclub.com).
Approximately 2ml of blood was collected from the cephalic vein of each dog and loaded into 4ml EDTA tubes and refrigerated at 4 ̊C as previously described (25)
Table 1 Total collected samples indicating sex, age and breed
Sex
|
Age
|
Breed
|
Male
|
Female
|
J
|
A
|
O
|
Local
|
Exotic
|
94
|
49
|
41
|
79
|
23
|
112
|
31
|
DNA extraction and characterization
Total DNA was extracted using the TanBead Automated DNA extractor. For extraction process optimization, two protocols were used; viral DNA extraction protocol and optipure protocol for blood DNA.300µl of whole blood were drawn from 6 randomly selected samples, and 10µl of Proteinase K was then added to each of the 6 samples. This mixture was then transferred to the TanBead extractor for total DNA extraction.DNA yield determined through agarose gel electrophoresis and Nanodrop Spectrophotometry indicated the better quantity and quality obtained from the optipure protocol. Therefore the rest of the samples were extracted using the optipure protocol. The extracted DNA was stored at -20̊C. (26)
Polymerase Chain Reaction (PCR)
To optimize PCR conditions for the screening PCR, total DNA was extracted according to the manufacturer’s instructions (using QIAGEN DNeasy kit® for blood and tissue using the nucleated tissue protocol) from blood drawn from a dog clinically diagnosed with babesiosis, the protocol was altered after adding lysis buffer, where the incubation temperature was set at 56 ̊C at 1000rpm for 12hrs. The extracted DNA sample was then diluted to 25ng/µl using triple distilled Milli-Q water (26, 27).
Gradient PCR was used to determine optimum diagnostic PCR conditions. Master mix was as follows 2x AccuPower master mix 5µl, pF 0.33µl, pR 0.33µl,g DNA 2.5µl,double distilled water 1.84µl for a total volume of 10.0µl.Diagnostic PCR conditions were as follows 95 ̊C for 5min,94 ̊C for 30sec,52 ̊C for 1min,72 ̊C for 1min,72 ̊C for 10min,15 ̊C ∞ .A 1.5% gel was prepared to run the PCR products at 135V for 30min. The results were visualized using a gel documentation system. (27)All the DNA samples were screened using the established diagnostic PCR conditions. The primers used were as outlined in table 2
Table 2 Primers used, their sequence, length, target gene and species
Primer
|
Oligo Sequence
|
Gene
|
Target Species
|
18S_rDNA_BTH_F
|
5’-CCT GMG ARA CGG CTA CCA CAT CT-3’ (23mer)
|
18S rDNA
|
Babesia genus
|
18S_rDNA_BTH_R
|
5’-TTG CGA CCA TAC TCC CCC CA-3’(20mer)
|
18S rDNA
|
Babesia genus
|
18S_rDNA_GF1
|
5’-GTC TTG TAA TTG GAA TGA TGG-3’(21mer)
|
18S rDNA
|
Babesia genus
|
18S_rDNA_GR2
|
5’-CCA AAG ACT TTG ATT TCT CTC-3’(21mer)
|
18S rDNA
|
Babesia genus
|
PCR products were visualized using agarose gel electrophoresis (28). The positive PCR products were purified using the QIAGEN quick gel purification kit according to the manufacturer’s instructions and the products submitted for sequencing.
Sequencing and analysis of PCR products
Sequencing was done by the Sanger Dideoxymethod at the International Livestock Research Institutes’ SegoliLab (29). The obtained sequences were analyzed using the CLC Genomics Workbench Version 20.0.2 where the sequences were trimmed, conflicts resolved using the forward and reverse sequences, and a consensus sequence generated (30). The consensus sequence was analyzed using the BLASTn program of the National Center for Biotechnology Information to identify closely related sequences on GenBank (31). Multiple sequence alignment and a phylogenetic tree were constructed using Geneious Prime 2020.1.2, Build 2020-04-07 08:42, Java Version 11.0.4+11(64bit) using the MUSCLE program ver 3.8.425 by Robert C.Edgar and Geneious tree builder. The genetic distance model used was Jukes-Cantor, while the tree build method was Neighbor joining; the number of bootstrap replicates was 1000. The following GenBank accession no’s were used in analyzing the sequences,AJ404608.1,HQ895984.1,HQ289870.1,AY150061.1,KT333456.1,LC331058.1,EU084681.1,MN067708.1,AB303076.1,MN625891.1,AY077719.1,DQ111765.1,LC331056.1,KY463434.1,DQ111760.1,JN982353.1,MH143395.1,MF040149.1,HM585429.1,AY618928.1,KF928958.1,KU204792.1 (32)