East Coast Fever Carrier Status and Theileria parva Breakthrough Strains in Recently ITM Vaccinated and Non-Vaccinated Cattle in Iganga District, Eastern Uganda

East Coast fever (ECF) is a tick-borne disease of cattle that hinders the development of the livestock industry in eastern, central and southern Africa. The ‘Muguga cocktail’ live vaccine, delivered by an infection and treatment method (ITM), remains the only immunisation strategy of controlling ECF. However, there are challenges of the live vaccine inducing ECF carrier status in immunised animals and the possibility of lack of protection from parasite strains that are antigenically different from the vaccine strains. In Uganda, there are insufficient data regarding the ECF carrier status and T. parva genetic diversity in vaccinated and associated non-vaccinated cattle to assess the effectiveness of ITM vaccination. Blood was collected from recently ECF vaccinated (98) and non-vaccinated (73) cattle from Iganga district in Eastern Uganda at 120 days post-vaccination. The p104 gene nested PCR was used to screen for T. parva DNA, 11 minisatellite and 3 microsatellite markers (SSR) were used for genotyping. Two minisatellite markers (MS7 and MS19) were used to determine whether ECF carrier status was due to the T. parva vaccine or local strains. The prevalence of T. parva based on p104 nPCR was 61.2% (60/98) (RR 2.234, 95% CI 1.49–3.35, p-value < 0.001) among recently vaccinated cattle and 27.4% (20/73) (RR 1.00) among associated non-vaccinated cattle. The Muguga cocktail vaccine strains were responsible for carrier status in 10 (58.8%) by MS7 and 11 (64.7%) by MS19 in vaccinated cattle. Genotypes of T. parva with different-sized alleles to the vaccine strains that could be potential ‘breakthroughs’ were detected in 2 (11.8%)) and 4 (23.5%) isolates from vaccinated cattle based on MS7 and MS19 minisatellite markers, respectively. Using 14 SSR markers, T. parva diversity was higher in vaccinated (Na = 2.214, Ne = 1.978, He = 0.465) than associated non-vaccinated (Na = 1.071, Ne = 1.048, He = 0.259) cattle. The principal component analysis (PCA) showed isolates from vaccinated cattle were closely related to those from non-vaccinated cattle. The analysis of molecular variance (AMOVA) revealed high genetic variation (96%) within T. parva isolates from vaccinated and non-vaccinated cattle but low variation (4%) between vaccinated and non-vaccinated cattle. This study reveals the role of ITM in inducing the carrier status and higher T. parva genetic diversity in vaccinated cattle. The low genetic variation between T. parva isolates in both vaccinated and non-vaccinated cattle may be suggestive of the protective role of vaccine strains against genetically related local strains in the study area.


Introduction
East Coast fever (ECF) is a tick-borne disease of cattle which is caused by a hemoprotozoan parasite called Theileria parva (T. parva). The parasite is transmitted by a brown ear tick (Rhipicephalus appendiculatus) and it is highly prevalent in eastern, central and southern Africa. East Coast fever (ECF) ranks first among the important tick-borne diseases of cattle in the region and causes high mortality, especially in exotic and cross-bred cattle, as well

Study Area
The study was conducted in Bulamagi and Nakigo sub-counties in Iganga district in the eastern region of Uganda ( Figure 1). The coordinates for Iganga district are: Latitude: 0 • 36 33.01 N, Longitude: 33 • 28 7.00 E. Districts along its boarders are Bugiri to the east, Namutumba to the northeast, Kaliro to the north, Kamuli to the northwest, Jinja to the west, and to the south lies Mayuge. The district has an average annual temperature of 22.3 • C and an average annual rainfall of 1313 mm, which are optimal for the survival of the livestock and disease vectors such as ticks. There are two relatively drier seasons (December to March and June to July) [18]. There is sufficient rainfall throughout the year to sustain man and animals except in very rare circumstances that sometimes lead to drought. T. parva genetic diversity was assessed from the vaccinated and non-vaccinated cattle. Data generated from this study provide additional information on the establishment and persistence of carrier status and genetic diversity of T. parva in the vaccinated and non-vaccinated cattle in an ECF endemic area. This is important for the use of ITM in the integrated control of ECF in endemic areas.

Study Area
The study was conducted in Bulamagi and Nakigo sub-counties in Iganga district in the eastern region of Uganda ( Figure 1). The coordinates for Iganga district are: Latitude: 0°36'33.01" N, Longitude: 33°28'7.00" E. Districts along its boarders are Bugiri to the east, Namutumba to the northeast, Kaliro to the north, Kamuli to the northwest, Jinja to the west, and to the south lies Mayuge. The district has an average annual temperature of 22.3 °C and an average annual rainfall of 1313 mm, which are optimal for the survival of the livestock and disease vectors such as ticks. There are two relatively drier seasons (December to March and June to July) [18]. There is sufficient rainfall throughout the year to sustain man and animals except in very rare circumstances that sometimes lead to drought.

Study Design
This was a cross-sectional study that involved sampling of cattle based on the ECF-ITM vaccination status, whereby both recently vaccinated (120 days post-vaccination using Muguga cocktail vaccine) and non-vaccinated cattle from the same farms. Vaccination status was confirmed from farmer records and special ear tags were put on the ECF vaccinated cattle. Fifteen cattle farms were selected from two sub-counties (Nakigo and Bulamagi) in Iganga district. Blood (5 mL) was drawn from the jugular vein into the EDTA vacutainer tube and placed in a cool box for short-term storage and, thereafter, transported on ice to the Molecular Biology Laboratory (MOBILA), College of Veterinary Medicine, Animal Resources and Biosecurity (COVAB) for laboratory analysis.

Study Design
This was a cross-sectional study that involved sampling of cattle based on the ECF-ITM vaccination status, whereby both recently vaccinated (120 days post-vaccination using Muguga cocktail vaccine) and non-vaccinated cattle from the same farms. Vaccination status was confirmed from farmer records and special ear tags were put on the ECF vaccinated cattle. Fifteen cattle farms were selected from two sub-counties (Nakigo and Bulamagi) in Iganga district. Blood (5 mL) was drawn from the jugular vein into the EDTA vacutainer tube and placed in a cool box for short-term storage and, thereafter, transported on ice to the Molecular Biology Laboratory (MOBILA), College of Veterinary Medicine, Animal Resources and Biosecurity (COVAB) for laboratory analysis.
Genomic DNA was extracted from cattle whole blood using the purelink TM genomic DNA mini kit (Invitrogen, Waltham, MA, USA). The DNA concentration and purity were determined using UV spectrophotometry (nanodrop) at 260-280 nm. The DNA was screened for presence of T. parva parasites using primers designed from p104 gene by nested PCR [19]. Positive p104 nPCR T. parva isolates were genotyped using fourteen satellite markers (SSRs): 11 mini-(MS) and 3 microsatellite (ms) markers; (MS7, MS19, MS3,  MS16, MS8, MS21, MS25, MS27, MS33, MS34, MS40, ms2, ms5, and ms7) [15]. The data generated were analysed to determine the prevalence, genetic diversity and persistence of carrier status of T. parva in recently vaccinated and associated non-vaccinated cattle in Iganga district. The MS19 and MS7 satellite markers were used to assess whether the carrier status was due to the vaccine or local parasite strains.

Recruitment and Vaccination of Animals
The animals were recruited into the study after a written informed consent by the cattle farmers. The inclusion criteria were based on animals ≥ 8 months old without fever (rectal temperature < 39.5 • C) and non-pregnant for the female animals. Cattle were ear-tagged and weighed. The immunisation procedure was carried out as described previously [8,20]. The immunised animals were injected with 1 mL of 100× dilution of MCL01 stabilate. The vaccine was inoculated subcutaneously in front of the right parotid lymph node and the animals were treated simultaneously with 30% long acting Oxytetracycline (Tetroxy LA, Bimeda, Dublin, Ireland) at a dose rate of 30 mg/kg body weight by deep intramuscular injection. The vaccine used was the trivalent formula known as Muguga Cocktail composed of Muguga, Kiambu-5 and Serengeti-transformed stocks [21]. The vaccine was purchased from SCOPEVET, (an authorised ECF vaccine importer/supplier in Uganda). The animals were monitored daily for 28 days post ITM vaccination for detection of clinical symptoms by the farmers and the Resident Veterinary Officers.

Detection of p104 Gene and Genotyping of Theileria parva Isolates Using Mini-and Microsatellite Markers by Nested PCR
Genomic DNA was extracted from 160 µL of EDTA vacutainer collected cattle blood using the purelink TM genomic DNA mini kit (Invtrogen, USA) according to manufacturer's instructions. The extracted DNA was stored at −20 • C until needed for T. parva screening and genotyping using nested PCR with p104 and SSR primers (Eurofins Genomics at GmbH, Viehmarktgassen 1B/Büro, AT-1030 Vienna, Austria). The nPCR for an invariable region of the p104 gene [19] was used to detect T. parva DNA in the extracted DNA test samples along with positive and negative controls. A negative control was a PCR reaction master mix with distilled water as DNA template. Theileria parva Muguga stock genomic DNA from International Livestock Research Institute (ILRI) was used as positive control.
For detection of p104 gene for Theileria parva, primary PCR amplification of the p104 gene generated a 496bp fragment using forward and reverse primers IL3231 (5'-ATTTAAGGAACCTGACGTGACTGC-3') and IL755 (5'-TAAGATGCCGACTATTAATGACACC-3'), respectively, as described by Skilton et al. [22]. Briefly, 2× Taq kappa with dye was used with the final volume of 10 µL; containing 5.0 µL 2X Taq kappa with dye, 0.25 µL of each forward and reverse primers, 2.5 µL of PCR grade nuclease-free water and 2.0 µL of the DNA template. The PCR conditions for amplifying the p104 gene in the primary PCR were as follows; initial denaturation at 94 • C for 5 min, followed by 40 cycles of denaturation at 94 • C for 1 min, primer annealing at 58 • C for 1 min, extension at 72 • C for 1 min. Final extension at 72 • C for 9 min and hold at 4 • C. The secondary PCR amplified a 277bp internal fragment located between bases 2784 and 3061 of the p104 gene using forward and reverse primers 5'-GGCCAAGGTCTCCTTCAGATTACG-3' and 5'-GTGGGTGTGTTTCCTCGTCATCTGC-3', respectively, as described by Odongo et al. [19]. The primary PCR product (1.0 µL) was used as a DNA template in a 10 µL reaction volume for the secondary PCR as above. The PCR conditions for amplifying the p104 gene in the secondary PCR were as described above except annealing temperature of 60 • C for 1 min. The secondary PCR products (5 µL) were checked on 2% agarose gel in Tris-acetate-EDTA (TAE) buffer at 125 V and 300 A for 40 min. Band size was determined using a 50 bp DNA in the secondary PCR were as described above except annealing temperature of 60 °C for 1 min. The secondary PCR products (5 µ L) were checked on 2% agarose gel in Tris-acetate-EDTA (TAE) buffer at 125 V and 300 A for 40 min. Band size was determined using a 50 bp DNA ladder (N32361, Biolabs). The DNA bands were visualised under UV light, photographed and documented ( Figure 2). For genotyping T. parva, positive p104 PCR samples and the Muguga cocktail vaccine DNA were used. The 3 micro-(ms2, ms5, ms7) and 11 minisatellite markers (MS3, MS5, MS7, MS16, MS19, MS2, MS25, MS27, MS33, MS34, MS40) [15] were used. Positive (T. parva Muguga stock DNA) and negative (distilled water) controls were run together with test samples. The MS7 and MS19 satellite markers were used to assess whether the T. parva carrier status was due to the vaccine or local strains [10,15,23].
The reaction mixture volumes used in primary PCR were as described above for detection of p104. The PCR conditions were set as follows; initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 65 °C for 1 min, final extension at 65 °C for 9 min and hold at 4 °C. The reaction mixture volumes used in secondary PCR were as described above in the nested p104 amplification. The secondary PCR conditions were set as described above in primary genotyping except using annealing temperature of 58 °C for 1 min. The secondary PCR products (5 µ L) were analysed as described above for p104 detection ( Figure 3).  [15] were used. Positive (T. parva Muguga stock DNA) and negative (distilled water) controls were run together with test samples. The MS7 and MS19 satellite markers were used to assess whether the T. parva carrier status was due to the vaccine or local strains [10,15,23].
The reaction mixture volumes used in primary PCR were as described above for detection of p104. The PCR conditions were set as follows; initial denaturation at 94 • C for 5 min, followed by 40 cycles of denaturation at 94 • C for 1 min, annealing at 55 • C for 1 min, extension at 65 • C for 1 min, final extension at 65 • C for 9 min and hold at 4 • C. The reaction mixture volumes used in secondary PCR were as described above in the nested p104 amplification. The secondary PCR conditions were set as described above in primary genotyping except using annealing temperature of 58 • C for 1 min. The secondary PCR products (5 µL) were analysed as described above for p104 detection ( Figure 3).

Data Analysis and Interpretation
Data generated from screening for T. parva DNA using p104 nPCR and genotyping using satellite markers (SSR) were entered and cleaned using Microsoft Excel. Descriptive statistics were computed at 95% confidence interval. Data from screening were analysed using Chi-square test to determine the association between outcome variable (T. parva positivity) and categorical variable (vaccination status) at statistical significance p < 0.05.
Data from genotyping were analysed using GenALEX software version 5 [24], which was used to calculate genetic diversity parameters. This included determining the mean number of alleles (Na), number of effective alleles (Ne) and expected heterozygosity (He). These parameters were used to determine parasite diversity (overall and within the parasite populations from vaccinated and non-vaccinated cattle). Principal Component Analysis (PCA) was used to determine the genetic relationships among T. parva isolates from Muguga cocktail vaccine, vaccinated and non-vaccinated cattle. Analysis of molecular variance (AMOVA) was also used to determine T. parva diversity by estimating the percentage variation within the individual population and between the two populations (vaccinated and non-vaccinated).

Data Analysis and Interpretation
Data generated from screening for T. parva DNA using p104 nPCR and genotyping using satellite markers (SSR) were entered and cleaned using Microsoft Excel. Descriptive statistics were computed at 95% confidence interval. Data from screening were analysed using Chi-square test to determine the association between outcome variable (T. parva positivity) and categorical variable (vaccination status) at statistical significance p < 0.05.
Data from genotyping were analysed using GenALEX software version 5 [24], which was used to calculate genetic diversity parameters. This included determining the mean number of alleles (Na), number of effective alleles (Ne) and expected heterozygosity (He). These parameters were used to determine parasite diversity (overall and within the parasite populations from vaccinated and non-vaccinated cattle). Principal Component Analysis (PCA) was used to determine the genetic relationships among T. parva isolates from Muguga cocktail vaccine, vaccinated and non-vaccinated cattle. Analysis of molecular variance (AMOVA) was also used to determine T. parva diversity by estimating the percentage variation within the individual population and between the two populations (vaccinated and non-vaccinated).
The detection of T. parva DNA in the vaccinated or non-vaccinated cattle blood was referred to as 'carrier' status since clinical ECF was not observed in the study animals. The appearance of parasite strain/genotype in the vaccinated cattle that was not similar to vaccine strains was considered as 'breakthrough'.

Farm and Farmer Characteristics
The cattle sampled (n = 171) were from fifteen farms from Iganga district in Eastern Uganda and they were kept under semi-intensive or zero grazing farming systems. The majority of the cattle sampled were 160 (93.6%) adults, 154 (90.1%) Friesian cross breed and 141 (82.5%) females (Table 1). The detection of T. parva DNA in the vaccinated or non-vaccinated cattle blood was referred to as 'carrier' status since clinical ECF was not observed in the study animals. The appearance of parasite strain/genotype in the vaccinated cattle that was not similar to vaccine strains was considered as 'breakthrough'.

Farm and Farmer Characteristics
The cattle sampled (n = 171) were from fifteen farms from Iganga district in Eastern Uganda and they were kept under semi-intensive or zero grazing farming systems. The majority of the cattle sampled were 160 (93.6%) adults, 154 (90.1%) Friesian cross breed and 141 (82.5%) females (Table 1).

Determination of the Source of Theileria parva Genotypes in ECF Vaccinated Cattle
In order to determine whether T. parva carrier status in recently vaccinated cattle was due to the vaccine or local strains, 80 positive p104 nPCR isolates were genotyped; however, only 32.5% (26/80) T. parva field isolates gave amplicons with both the MS7 and MS19. Using MS7 minisatellite marker, 58.8% (10/17) of T. parva DNA from vaccinated cattle amplified alleles of the same size as that amplified by the Muguga vaccine DNA (300 bp). The extracted DNA samples from non-vaccinated cattle (44.4% (4/9)) amplified alleles of the same size as that amplified by the Muguga cocktail vaccine (300 bp) and 55.6% (5/9) of the non-vaccinated cattle showed an allele of 150bp, which also appeared in 29.4% (5/17) of DNA from vaccinated cattle. The allele band size (150 bp) was predominant in local strains and absent in the cocktail vaccine. Some isolates from vaccinated cattle 11.8% (2/17) amplified an allele of 280 bp, which was not amplified in the majority of the isolates from non-vaccinated cattle and also not detected in the Muguga cocktail vaccine strains. The 280 bp allele, when detected in vaccinated cattle, was assumed to be a breakthrough strain. Two alleles (200 bp and 380 bp) were amplified from the cocktail vaccine DNA but were neither amplified from isolates from vaccinated nor non-vaccinated cattle. Many isolates from vaccinated cattle (47.1% (8/17)) had more than one allele, indicative of multiple infections by many parasite strains. A total of 33.3% (3/9) of isolates from non-vaccinated cattle also had two alleles each (Figure 3).

Determination of the Genetic Relationship between Theileria parva Isolates from Vaccinated and Non-Vaccinated Cattle
In the principal component analysis (PCA) plot, there was no distinct clustering as the parasite alleles scattered throughout the plot. However, most of the parasite alleles

Determination of the Genetic Relationship between Theileria parva Isolates from Vaccinated and Non-Vaccinated Cattle
In the principal component analysis (PCA) plot, there was no distinct clustering as the parasite alleles scattered throughout the plot. However, most of the parasite alleles from vaccinated and non-vaccinated cattle appeared in the two upper and lower right quadrants. The lower left quadrant contains alleles from vaccinated cattle and Muguga cocktail vaccine ( Figure 5).

Determination of the Genetic Variation of Theileria parva Parasites from Vaccinated and Non-Vaccinated Cattle Populations
Analysis of molecular variance (AMOVA) calculated from the two parasite populations revealed a large percentage of genetic variation (96%) within individual populations with only 4% being explained by differences among populations (Table 4).

Determination of the Genetic Variation of Theileria parva Parasites from Vaccinated and Non-Vaccinated Cattle Populations
Analysis of molecular variance (AMOVA) calculated from the two parasite populations revealed a large percentage of genetic variation (96%) within individual populations with only 4% being explained by differences among populations (Table 4).

Discussion
This study investigated the carrier status and genetic diversity of T. parva in recently vaccinated cattle of 120 days post vaccination. There was an overall T. parva prevalence of 46.8% in cattle sampled from two sub-counties in Iganga district. The prevalence was higher in recently Muguga cocktail-vaccinated cattle (61.2%) than non-vaccinated cattle (27.4%). Previous studies conducted in Tanzania also reported similar results where prevalence of T. parva was higher in vaccinated cattle than non-vaccinated cattle [25,26]. More than half of the recently vaccinated cattle became carriers of T. parva, justifying the importance of ITM since the majority of the vaccinated cattle become carriers and remain immune to ECF [27]. The persistence of carrier state for up to 120 days post vaccination was probably a combined effect of vaccination and the continuous tick challenge to cattle under natural field conditions exposure. This is confirmed by the fact that tick exposure plays a major role in form of an incremental booster effect on the ITM-induced immunity [25].
The T. parva carrier status was not detected in all the recently vaccinated cattle. Failure to detect the carrier state in all vaccinated cattle, however, does not necessarily mean vaccine failure or lack of immunity in an animal, because some cattle after vaccination may clear the parasite and remain immune in absence of a carrier state, a condition known as sterile immunity [10,28]. It is also possible that the parasite may be present in other tissues such as lymph nodes, other than blood [28]. However, vaccination failure can also occur whereby a high dose of long-acting Oxytetracycline is administered during the ITM vaccination and may clear all the infection. Alternatively, the carrier status may wane when not boosted by infected ticks under natural tick challenge. None of the vaccinated cattle showed clinical signs of ECF disease, implying that the vaccine protected them. In the case of the non-vaccinated cattle, the natural infection may have induced a carrier status that elicits innate or natural immunity. The induced carrier status by natural infection is boosted and maintained by feeding infected ticks to the animals, thus providing natural immune protection. In addition, the improved farm management could have influenced the outcome of the lack of clinical symptoms, since farmers adhered to the appropriate use of acaricides to control the ticks during the study period, which may have kept the parasitaemia low. However, ECF is known to be a prevalent disease in the study area. Screening of non-vaccinated cattle using a nested p104 PCR revealed T. parva prevalence of 27.4% (20/73), which was much lower than vaccinated cattle. The non-vaccinated cattle could be carriers of the local strains of T. parva circulating in the field or they could be carriers of vaccine strains since it is possible for the local tick population to become infected with the vaccine parasite strains from vaccinated animals with resultant transmission to the non-vaccinated cattle which share grazing grounds [10]. There was, however, a significant difference in proportion of carriers between vaccinated and non-vaccinated cattle whereby prevalence of T. parva was higher in vaccinated cattle than non-vaccinated cattle, which confirms the impact of live vaccination in inducting a carrier state [26].
In order to answer the question of which strains of T. parva persisted in the cattle population after immunisation with Muguga cocktail vaccine, polymorphisms in the minisatellite markers MS7 and MS19 were studied. The MS7 and MS19 SSR markers were chosen because of their high sensitivity and their ability to differentiate between genotypes of T. parva from vaccine and field strains [16]. Some of the alleles which occurred in the Muguga cocktail vaccine DNA were detected in both vaccinated and non-vaccinated cattle. The appearance of the vaccine alleles in non-vaccinated cattle implies that such alleles may have been transmitted by ticks from vaccinated to non-vaccinated cattle or some of the local strains of T. parva circulating in the cattle population are similar to the vaccine strains. There were some alleles with band sizes of 200 bp and 380 bp which occurred in the Muguga vaccine with MS7 primers but were not detected in isolates from vaccinated cattle. These alleles appearing in the vaccine but were absent in the vaccinated cattle could be T. parva Serengeti-transformed stock and Muguga stocks since the Muguga vaccine is a cocktail of three main stocks; Muguga, Kiambu 5 and Serengeti-transformed [15]. The Muguga and Serengeti-transformed stocks induce a short-term carrier state of up to 87 days post vaccination, whereas Kiambu 5 induces a long-term carrier state [10,13]. The predominant alleles that were occurring in the majority of the non-vaccinated and in some of the vaccinated cattle isolates that are different from the Mugaga vaccine strains could possibly be local strains of T. parva circulating in the field. A study conducted by Oura et al. [10] also found that the vaccinated cattle can become infected with some local strains of T. parva in the field but these do not result in overt disease. However, there were alleles occurring in some isolates from vaccinated cattle that were neither observed in the vaccine nor from non-vaccinated cattle isolates, which could be possible 'breakthrough' strains [13].
Genotyping of T. parva isolates gave 3-5 alleles from vaccinated and 2-4 alleles from non-vaccinated cattle. The presence of numerous parasite allelic components reflects great antigenic heterogeneity and hence broadens the protection induced by vaccination. Individual parasite DNA isolates, however, carried either only one or two alleles each. This reflects a phenomenon that only a limited subset of T. parva genotypes can be transmitted by ticks to cattle, although T. parva genotypes circulating in the field are highly diverse [29]. The mean number of different alleles, mean number of effective alleles and expected heterozygosity were used to compare T. parva diversity among parasite populations. All these parameters were high in vaccinated (mean number of different alleles, 2.214; mean number of effective alleles, 1.978; expected heterozygosity, 0.465) than non-vaccinated cattle (mean number of different alleles, 1.071; mean number of effective alleles, 1.048; expected heterozygosity, 0.259). This implies that T. parva parasite diversity in recently vaccinated cattle was higher than the diversity in non-vaccinated cattle. This confirms the positive role of ITM of vaccination in increasing the diversity of T. parva parasites, which may be enhanced through influence of sexual recombination and continuous tick challenge, which may lead to wider immune protection [30].
The principal component analysis (PCA) depicted close genetic relatedness of T. parva parasite isolates from recently vaccinated and non-vaccinated cattle since they always scattered together. This shows that there was sharing of strains between vaccinated and non-vaccinated cattle. This has been confirmed as a possible scenario in the field where ticks aid in transferring T. parva parasites between cattle that share common grazing ground [10,26]. However, isolates from vaccinated cattle were more closely related to the strains from Muguga cocktail vaccine, as shown by the clustering in the lower left quadrat ( Figure 5). The low genetic variation of 4% among the two populations also confirms that the two populations shared similar genetic composition. The parasite isolates scattered in all the four quadrants, implying that the T. parva parasites were genetically diverse in the field, which could be induced by genetic recombination between field parasites and vaccine parasites [30]. The principal component analysis plot findings were consistent with the high level of variation existing within individual parasite isolates in a population (96%), implying that there was high genetic diversity in each individual population. This is similar to findings by Magulu et al. [26].
During genotyping of field isolates with minisatellite markers MS7 and MS19 to determine whether T. parva carrier status was due to vaccine strains or due to field strains, a Muguga cocktail vaccine DNA (Serial no. ECF MCL 0202 CTT BD DEC 14) was used as reference control. However, it was difficult to point out exactly which strain of T. parva was causing carrier status among the 58.8% (MS7) and 64.7% (MS19) of the vaccinated cattle isolates which were carriers of vaccine strain. This is because a Muguga cocktail vaccine is composed of three stocks of T. parva; Muguga stock, Kiambu 5 stock and Serengetitransformed stock. However, studies have shown that only Kiambu 5 stock alleles can be detected in vaccinated cattle isolates by PCR after 87 days: therefore, its alleles were the only ones expected. For clarity, it may have been best to use individual vaccine cell line stock components as reference controls. However, this was impended by limited finances to purchase the individual vaccine cell lines, as the Muguga cocktail vaccine used was easy to access since the project was involved in the vaccination exercise of cattle in Iganga district. In addition, there is a need to isolate the antigenic region from non-vaccine T. parva strains detected in vaccinated and non-vaccinated cattle and sequence to determine their genetic make-up and compare with the known strains of T. parva to ascertain the possibility of 'breakthrough' strains. Additionally, other tests such as serology (antibody test) should be performed to ascertain whether the strains causing carrier status among vaccinated cattle could be from previous infection, thus carriers already probably existed if the cattle were previously vaccinated, especially for cattle farmers that had not documented properly.

Conclusions
The study revealed that the prevalence of T. parva was high in both vaccinated (61.2%) and non-vaccinated (27.4%) cattle at 120 days post vaccination. This was brought about by the carrier status of T. parva following Infection and treatment method (ITM) of vaccination using Muguga cocktail vaccine. The T. parva carrier status provided immunity to a high number of vaccinated and non-vaccinated cattle since no clinical symptoms of disease were observed. The T. parva Muguga cocktail vaccine strains were responsible for carrier status in the majority of the recently vaccinated cattle; 58.8% (MS7) and 64.7% (MS19). The non-vaccine strains causing carrier status in vaccinated cattle could be field strains or breakthrough strains. In addition, there were peculiar strains that appeared in 11.8% (MS7) and 23.5% (MS19) of vaccinated cattle, which could likely be breakthrough strains. The high genetic diversity of T. parva was due to continuous natural tick exposure in the field, leading to parasite strain recombination generating more genotypes, which brings about the high parasite allelic diversity in vaccinated and non-vaccinated cattle that share common grazing ground. Informed Consent Statement: Written informed consent were obtained from the cattle farmers prior to commencement of the study.

Data Availability Statement:
The datasets used to analyse during current study are available from the corresponding author on request.