Population genetic analysis and sub-structuring of Theileria parva in the northern and eastern parts of Zambia
- Walter Muleya†1, 2,
- Boniface Namangala†3,
- Martin Simuunza†4,
- Ryo Nakao†5,
- Noboru Inoue†6,
- Takashi Kimura†1,
- Kimihito Ito†7, 8,
- Chihiro Sugimoto†5, 8 and
- Hirofumi Sawa1, 8Email author
© Muleya et al.; licensee BioMed Central Ltd. 2012
Received: 5 May 2012
Accepted: 1 October 2012
Published: 12 November 2012
Theileriosis, caused by Theileria parva, is an economically important disease in Africa. It is a major constraint to the development of the livestock industry in some parts of eastern, central and southern Africa. In Zambia, theileriosis causes losses of up to 10,000 cattle annually.
Cattle blood samples were collected for genetic analysis of Theileria parva from Isoka and Petauke districts in Zambia. Microsatellite analysis was then performed on all Theileria parva positive samples for PCR using a panel of 9 microsatellite markers. Microsatellite data was analyzed using microsatellite toolkit, GenAlEx ver. 6, Fstat ver. 22.214.171.124, and LIAN computer softwares.
The combined percentage of positive samples in both districts determined by PCR using the p104 gene primers was 54.9% (95% CI: 46.7 – 63.1%, 78/142), while in each district, it was 44.8% (95% CI: 34.8 – 54.8%) and 76.1% (95% CI = 63.9 – 88.4%) for Isoka and Petauke districts, respectively. We analyzed the population genetic structure of Theileria parva from a total of 61 samples (33 from Isoka and 28 from Petauke) using a panel of 9 microsatellite markers encompassing the 4 chromosomes of Theileria parva. Wright’s F index (FST = 0.178) showed significant differentiation between the Isoka and Petauke populations. Linkage disequilibrium was observed when populations from both districts were treated as a single population. When analyzed separately, linkage disequilibrium was observed in Kanyelele and Kalembe areas in Isoka district, Isoka district overall and in Petauke district. Petauke district had a higher multiplicity of infection than Isoka district.
Population genetic analyses of Theileria parva from Isoka and Petauke districts showed a low level of genotype exchange between the districts, but a high level of genetic diversity within each district population, implying genetic and geographic sub-structuring between the districts. The sub-structuring observed, along with the lack of panmixia in the populations, could have been due to low transmission levels at the time of sampling. However, the Isoka population was less diverse than the Petauke population.
Theileriosis is an economically important disease of cattle in eastern, central and southern Africa. The disease, caused by the protozoan haemoparasite Theileria parva (T. parva), is transmitted by the 3-host tick Rhipicephalus appendiculatus. The severity of the disease manifests differently in various breeds of cattle, with the zebu cattle (Bos indicus) being more resistant than the exotic breeds (Bos taurus) against both the parasite  and the vector [3, 4]. Theileriosis causes high levels of mortality in taurine breeds  and both high morbidity and mortality in indigenous 1–6 month old calves (Bos indicus). Full-scale epidemics affecting all age groups of indigenous breeds may occur , resulting in reduction in productivity. The high level of mortality in taurine breeds impedes the introduction of highly productive exotic breeds in endemic areas, further preventing the improvement of cattle production in the affected areas. Theileriosis is thus a major constraint to the development of the livestock industry in the eastern, central and southern parts of Africa.
T. parva is mainly haploid during its life cycle, except for the polyploidy schizont stage and the diploid zygote and kinete stages . Briefly, T. parva male and female gametes in the tick gut fuse to form short-lived diploid zygotes which then develop into motile kinetes  and migrate to the tick salivary glands where they divide meiotically into sporozoites [10, 12]. The sporozoites, once introduced into cattle by the feeding tick, rapidly invade leucocytes, differentiating into intralymphocytic schizonts with a certain proportion undergoing merogony giving rise to uni-nucleate merozoites [10, 13]. The rupture of lymphocytes liberates the merozoites into the blood stream where they develop into intraerythrocytic piroplasms, the stage that is ingested by the tick vector during a blood meal [10, 13]. Genetic exchange has been shown to occur when 2 or more stocks of T. parva are used to infect the tick vector .
Studies on the population structures of various protozoan parasite species [15–21] have shown clonal (Trypanosoma cruzi, Toxoplasma gondii and Cryptosporidium hominis) [22–24], panmixic (Cryptosporidium parvum)  and epidemic (Crytosporiduim parvum)  population structures. On the other hand, Trypanosoma brucei and Plasmodium falciparum population structures have shown dependence on host specificity and transmission intensity [26, 27]. In general, the conclusion from these studies is that despite the presence of an obligatory sexual phase in their life cycle, a number of factors determine the population structures of different protozoan parasitic species.
Micro- and mini-satellite markers have been used to genotype several species of apicomplexan parasites and their vectors, revealing different population structures [18, 21, 26–30]. For example, a study on the population structure of T. parva in Uganda reported a mixture of genotypes in many isolates and linkage disequilibrium (LD) in 3 populations isolated from different areas . When multiple isolates with identical genotypes were treated as a single isolate, the presence of an epidemic structure was seen in 2 of the populations, suggesting an intermediate between extreme clonality and panmixia .
A crucial and important aspect in population genetic studies is to determine the effect of random and non-random mating on the population structures of disease-causing agents and consequently on the epidemiology of the diseases . Information on the genetic exchange in T. parva populations has practical implications in disease control and prevention. For instance, populations with a high degree of genetic diversity arise when high levels of recombination occur. This information is very important in vaccine development as it is easier to develop a vaccine against a clonal pathogen than a highly diverse pathogen. A panel of 9 polymorphic micro-satellite markers was used to genotype T. parva positive cattle blood DNA in order to answer the following questions: (i) what is the population structure of the T. parva in eastern and northern Zambia? (ii) Does gene flow occur between T. parva populations sampled from each area? (iii) Do the T. parva populations from the sampling areas consist of a single or multiple distinct populations? To our knowledge, this is the first study on the population genetics of T. parva in Zambia.
Sample collection and DNA preparation
This study received ethical clearance for collection of animal blood from the Biomedical Research Ethics Committee, University of Zambia and from the Department of Veterinary and Livestock Development, Eastern Province, Zambia. The recombinant DNA experiments were approved by Hokkaido University.
Panel of microsatellite markers used to genotype Theileria parva samples from Isoka and Petauke districts
Amplicon size (bp)
Used in final analysis
Oura et al., 2003 
Katzer et al., 2010 
Katzer et al., 2006 
Oura et al., 2003 
Katzer et al., 2010 
Katzer et al., 2010 
Katzer et al., 2010 
Katzer et al., 2010 
Katzer et al., 2010 
Katzer et al., 2010 
Katzer et al., 2010 
Katzer et al., 2006 
Katzer et al., 2006 
Katzer et al., 2006 
Katzer et al., 2006 
Katzer et al., 2006
T. parva screening
Whole blood DNA field samples were screened for T. parva DNA by using T. parva-specific p104 gene primers . Template DNA (1μL) was amplified in a 20 μL reaction mixture as prescribed by the manufacturer using ExTaq polymerase (Takara, Tokyo, Japan). The PCR conditions were as follows: denaturation at 95°C for 5 minutes; 35 cycles at 95°C for 1 minute, 63°C for 30 seconds and 72°C for 1 minute, followed by a final extension step of 5 minutes at 72°C. The amplified products were analyzed on 2% ethidium bromide pre-stained agarose gel.
PCR amplification and analysis of microsatellite loci
PCR was performed using primers (Table 1) designed to amplify each of the 16 repeat regions on each of the T. parva stocks isolated from different geographical areas. The forward primer in each primer marker set was fluorescently labeled. The 20 μL PCR mixture used comprised of 10 ng of template DNA, 10 μL of 2x Amplitaq Gold master mix (Invitrogen, CA), 0.25 μM of each primer and distilled water. The PCR conditions were as follows: denaturation at 95°C for 5 minutes; 35 cycles at 95°C for 15 seconds, 50 - 58°C for 60 seconds and 72°C for 1 minute, followed by a final extension step of 5 minutes at 72°C. The amplified products were observed on 1.5% ethidium bromide pre-stained agarose gels to determine the success of PCR amplification. To achieve high genotyping resolution of field samples, microsatellite PCR products were denatured and then capillary electrophoresed in an ABI 3130 genetic analyzer (Applied Biosystems, CA). DNA fragment sizes were analyzed relative to the ROX-labeled GS600 LIZ size standard (Applied Biosystems) using Gene Mapper software (Applied Biosystems). This facilitated the resolution of multiple products with 1 base pair (bp) difference in a single reaction. Multiple products from a single PCR reaction indicated the presence of mixed genotypes. The output data from the genetic analyzer were provided as the area under the peak of each allele (quantitative measurement), with the predominant allele possessing the greatest peak area. In this way, the predominant allele at each locus was identified for each sample, and this data was combined to generate a multi-locus genotype (MLG) representing the most abundant genotype in each sample. Only the alleles with the prescribed base pair range were used to generate the MLG and samples from the same area were electrophoresed and genescaned on the same plate. Separate PCRs were also carried out for different regions.
An allele sharing co-efficient  in Excel microsatellite toolkit (http://animalgenomics.ucd.i.e./sdepark/ms-toolkit/) was used for the similarity comparison of the MLGs . Principal component analysis (PCA) was used for similarity analysis. A similarity matrix was constructed and used to construct PCA using the Excel plug-in software GenAIEx6 (http://www.anu.edu.au/BoZo/GenAIEx/) . FSTAT computer package version 126.96.36.199 was used to calculate estimates of F statistics for population genetic analysis (http://www2.unil.ch/popgen/softwares/fstat.htm). LIAN (http://adenine.biz.fh-weihenstephan.de/lian/) was used to test the null hypothesis of linkage equilibrium by calculating a quantification of linkage equilibrium/linkage disequilibrium called the standardized index of association (I A S ) . The statistical independence of alleles at all pairwise combinations of loci under study characterizes linkage equilibrium (LE) and this independent assortment was initially tested by LIAN by determining the number of loci at which each pair of MLGs differs. The mis-match values from this distribution were then used to calculate the variance (VD) which was then compared to the variance expected (VE) for LE. Monte Carlo (MC) computer simulation was used to test the null hypothesis that VD = VE. The computer software calculates a 95% confidence limit L (LMC). When VD is greater than L, the null hypothesis of LE is discarded.
Multiplicity of infection
The majority of the samples in this study possessed several alleles at one or more loci, representing a mixed infection. The mean number of alleles for the nine loci in each sample was calculated and this index value represented the multiplicity of infection within each sample. The overall mean for the index values for each sample was then calculated to provide the multiplicity of infection for each region.
The screening of cattle blood samples (n=142) using the p104 gene primers showed a combined T. parva positive percentage of 54.9% (95% CI: 46.7–63.1%, 78/142) from both districts. The percentage of positive samples within each district was 44.8% (95% CI: 34.8–54.8%) (23 from Kanyelele and 20 from Kalembe, 43/96) and 76.1% (95% CI: 63.9–88.4%, 35/46) for Isoka and Petauke (Saukani area), respectively.
Marker diversity and allelic variation
A panel of 9 polymorphic microsatellite markers out of the initial 16 markers (Table 1), representing the 4 chromosomes of T. parva, was used to genotype 61 T. parva positive samples, that is 33 (14 from Kalembe and 19 from Kanyelele areas) from Isoka district and 28 from Petauke district (Saukani area). Seven markers that either failed to produce signals on gel electrophoresis or exhibited reduced polymorphism were excluded from the final analysis (Table 1). Seventeen samples were excluded from the final analysis because most of the markers failed to produce signals in them on gel electrophoresis. The reason for the failure of amplification of the target regions in these samples (n=17) was probably due to the poor quality of the DNA.
Allelic variation among Theileria parva from Petauke and Isoka districts of Zambia
Alleles within population
Similarity analysis between the Petauke and Isoka populations
Population diversity and differentiation
Population genetic analyses of Theileria parva from Petauke and Isoka districts of Zambia
Mean numbers genotypes/locus
I A S
To measure the levels of genetic differentiation, Wright’s F index was calculated between Isoka district and Petauke district populations, for the Isoka sub-populations and also within Petauke district (Table 3). An FST value of 0.178 was observed between Isoka and Petauke district populations (Table 3), showing significant genetic differentiation. The FST value for the Isoka populations (Kanyelele and Kalembe) was 0.049, implying moderate differentiation between the 2 populations (Table 3). The FST value for Petauke (Figure 3C) was 0.154, showing significant differentiation.
In order to determine whether the parasite populations within the two districts comprised a single panmictic population with a high degree of genetic exchange, the level of LE of the alleles at pairs of loci was measured using the standard index of association (I A S ). The I A S measures the association between alleles at pairs of loci, with I A S values close to 0 or negative indicating panmixia and those significantly greater than 0 indicating non-panmixia . The variance of pairwise differences (VD) between the data and that predicted for panmixia (VE) and L, the 95% confidence interval for VD relative to the null hypothesis, were calculated in order to test the hypothesis of panmixia. Using this analytical method, when the VD value exceeds the L value, LD is indicated and the null hypothesis of panmixia is discarded. When the VD is less than L, LE is indicated and the null hypothesis of panmixia is accepted. When Isoka and Petauke samples were treated as a single population, a I A S value of 0.078 (p< 0.001) and a VD value greater than the value of L was obtained, suggesting LD (Table 3). To test the hypothesis of geographical sub-structuring, the I A S , VD, and L values were calculated separately for samples from each district. The overall I A S value for Isoka district was 0.104 (p< 0.001) and the VD value was greater than the L Value, indicating a state of LD. The I A S values for Kanyelele and Kalembe populations within Isoka were 0.075 (p< 0.001) and 0.243 (p< 0.001), respectively, and both areas showed a VD value greater than the L value, suggesting LD (Table 3). An overall I A S value of 0.027 (p< 0.015) and a VD value greater than the L value indicating LD were obtained for the Petauke population.
Multiplicity of infection
Multiplicity of infection in Petauke and Isoka districts
Petauke and Isoka districts (overall)
In order to establish effective control measures as well as to assess the effectiveness of the current control measures, information on the population structure of T. parva with regard to its epidemiology is important. In this study, we analyzed samples from Kanyelele and Kalembe areas in Isoka district (NP) and from Saukani area in Petauke district (EP) of Zambia, which are approximately 600 km (straight line distance) apart and are separated by the North Luangwa, South Luangwa and Lukusuzi National Parks (Figure 1). To achieve this, we employed micro-satellite analysis, an effective way of studying population structures of a wide range of species. Micro-satellite analysis enables direct genotyping of parasite isolates directly from host blood samples by using specific primers. Recently, a panel of polymorphic micro- and mini-satellite markers for T. parva was identified [32–34]. A panel of 16 of these microsatellite markers was initially chosen and only 9 were used in the final analysis (Table 1). All the markers (locus pairs) on the same chromosome were over 182 kbp apart. It is therefore unlikely that any of the markers could have been physically linked. To perform the population genetic study of T. parva, a haploid organism, an MLG was constructed for each sample, by assigning a single predominant allele for each marker at each locus for each sample. This method of selecting the most predominant allele at each locus from mixed infections is currently fairly standard although it has limitations [18, 21, 25]. There are several potential short comings of selecting predominant alleles from mixed populations to form an MLG: (i) each particular marker does not always amplify the predominant allele from all strains (ii) the same strain is not always amplified by a particular marker in all the samples, (iii) target regions in different strains that have the highest sequence homology with markers are easily amplified whether they are representative of the predominant strain or not and (iv) all markers in the selected panel of markers most likely do not amplify the same strain in different samples. To avoid these short falls in mixed infections would involve cloning out samples or to only chose samples with only a single allele and then proceed with analysis and generation of an MLG. In this study however, we used a quantitative method to generate an MLG and by so doing, we assume that this MLG presented the closest representative of the dominant strains across the whole sample size.
Allele frequencies at each locus from the Isoka and Petauke populations showed a high number of unique alleles in each population (Figure 2). The data obtained using markers MS48 and MS53 indicated the possibility of genetic differences between Isoka and Petauke populations as evidenced by the complete lack of shared alleles at these 2 sites (Figure 2A). The separation of alleles on loci MS48 and MS53 might also be due to artifacts and as such the data on these loci should be treated with caution; however analysis of the overall data with and without loci MS48 and MS53 produced similar results (data not shown). The increased number of unique alleles versus shared alleles observed when Isoka and Petauke populations were treated as a single population (Figure 2B) suggests a state of genetic and geographical sub-structuring. Genetic and geographical sub-structuring was also observed on PCA where samples from Isoka and Petauke districts clustered separately (Figure 3A). A few samples (n=3) from Isoka district occupied the same quadrants as those from Petauke (Figure 3A). This state of genetic and geographical sub-structuring indicated by allele frequencies (Figure 2C) and PCA (Figure 3A) was further confirmed by the significant differentiation (FST = 0.178) observed between the population from Isoka and Petauke districts. Furthermore, a state of LD with a I A S value greater than 0 was also observed when the Isoka and Petauke populations were treated as a single population (Table 3), indicating the absence of random mating between T. parva from the 2 populations and consolidating the state of genetic and geographical sub-structuring. The population from Isoka exhibited less diversity than that from Petauke. These observations therefore, suggests that the parasite populations from these 2 areas, while comprising a similar number of genotypes per locus, exist as separate populations because there is little or no movement of animals between these 2 districts, which in turn is caused by their separation by physical geographical barriers i.e. the national parks (Figure 1). These findings are in agreement with a study on a T. parva population wherein geographical sub-structuring was observed between the population from 2 areas separated by a distance of 300 km and a lake .
The population from Kanyelele occupied all four quadrants while that from Kalembe only occupied three quadrants (Figure 3B). Although the Kanyelele population was more diverse than the Kalembe population in Isoka district, only moderate genetic differentiation (FST = 0.049) was observed between the 2 populations (Table 3). This was indicative of the existence of similar genotypes of T. parva in both areas. However, the ability of the parasites to randomly mate was restricted as shown from the state of LD, both at the district level and within each sub-population (Table 3). In the Petauke population, significant genetic differentiation (FST = 0.154) and a state of LD was also observed. This state of LD in Isoka (overall and in sub-populations) and Petauke might be due to the lack of mixing or movement of animals resulting in a lack of gene flow between the populations that can likely be attributed to the relative isolation of the sample areas from each other and the resulting restricted circulation of genotypes. Despite Kanyelele and Kalembe being far apart, animals from the two areas tend to share grazing grounds during the dry period because of the scarcity of pastures and thus it can be hypothesized that the sharing of grazing grounds allows the introduction of genotypes indigenous to one area into another area. However, due to the availability and close proximity of moderate vegetation for animal grazing during the month of May and the preceding months, it is unlikely that animals from Kanyelele and Kalembe areas would have mixed or shared grazing grounds as they will tend to graze pastures closer to their villages. This lack of mixing of animals would have prevented the introduction of genotypes from one area into another and vice-versa, hence the state of restrictive circulation. The tick vector population is also low at this particular time of the year, thus reducing the challenge levels of infection (i.e. low levels of transmission). It can further be hypothesized that the low challenge levels of infection is also likely to give rise to the presence of fewer genotypes of T. parva which may result in the state of LD and non-panmixia observed in the majority of areas/regions. This is in agreement with a report in which Plasmodium falciparum populations showed strong LD, extensive population differentiation and low diversity in regions of low transmission . The state of LD in these populations might also be due to the presence of epidemic strains . A large sample size, coupled with tick vector ecology data of a wider geographical area in Petauke and Isoka districts, is required to completely validate the LD states observed in this study.
The majority of samples analyzed in this study comprised mixed infections, with Petauke district showing a higher multiplicity of infection than Isoka district (Table 4). Within Isoka district, Kanyelele area also showed a higher multiplicity of infection than Kalembe area (Table 4). Several reasons may be advanced for this situation including (i) transmission intensity reflected by the tick burden on cattle, (ii) the level of infection of the parasite in the ticks, (iii) cattle host factors such as age and breed, (iv) farming systems and (v) the geography of the respective regions. However, this data was inconclusive and as such the effect of these factors could not be further investigated.
The results of this study indicate that the T. parva population from Isoka district is distinctively different from that of Petauke district, with significant gene diversity in each population. There was no evidence of genetic exchange among the populations from the 2 districts. Additionally, these results show that the populations from Kanyelele and Kalembe areas in Isoka district comprised similar genotypes although panmixia of parasites between the 2 areas could not be demonstrated. The sub-structuring and population structure of the T. parva populations observed in each district could be due to the restrictive circulation of parasites as a result of the lack of mixing of animals from different regions and/or the low transmission levels of T. parva in the respective areas within each district, which may be due to the reduced population of tick vectors at the time of sampling.
We thank Ms. Yamanouchi and Ms. Ohnuma, Research Center for Zoonosis Control, Hokkaido University, for technical assistance. This study was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT); the Ministry of Health, Labor and Welfare, Japan; the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID), MEXT Japan. We also wish to thank the farmers and field veterinary officers in Isoka and Petauke including the Eastern province veterinary officer for their cooperation.
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