Molecular detection and characterisation of protozoan and rickettsial pathogens in ticks from cattle in the pastoral area of Karamoja, Uganda.

Ticks and tick-borne diseases (TBDs) significantly affect cattle production and the livelihoods of communities in pastoralist areas. Data on protozoan and rickettsial pathogens in ticks infesting cattle in Uganda is scanty; while it is an indicator of the likelihood of disease transmission and occurrence. A cross-sectional study was conducted amongst cattle in the Karamoja Region, northeastern Uganda, from July through September 2017, to determine the tick species diversity, identify protozoan and rickettsial pathogens in the ticks, and characterise pathogenic species by sequence and phylogenetic analyses. About 50 % of the ticks detected from each predilection site on each animal were collected from 100 purposively-selected cattle from 20 randomly-selected herds. Twelve tick species belonging to the genera Amblyomma, Rhipicephalus and Hyalomma were identified, the most abundant being Amblyomma lepidum (93.9 %), followed by Amblyomma variegatum (2.0 %) and Rhipicephalus evertsi evertsi (1.0 %). Tick species that have not been reported in recent studies amongst cattle in Uganda were found, namely Rhipicephalus pravus, Rhipicephalus praetextatus and Rhipicephalus turanicus. The ticks were grouped into 40 pools, by species and location, and the reverse line blot (RLB) hybridisation assay was used to detect pathogens from the ticks. The most frequently detected tick-borne parasites were Theileria mutans, Theileria velifera and Theileria parva, each observed in 25 % (10/40) of the tick pools. Tick-borne pathogens, namely Babesia rossi, Babesia microti and Theileria sp. (sable) that are not common to, or not known to infect, cattle were identified from ticks. The gene encoding Ehrlichia ruminantium pCS20 region, the Ehrlichia and Anaplasma 16S rRNA gene, and T. parva p67 sporozoite antigen gene were amplified, cloned and sequenced. Seven novel E. ruminantium pCS20 variants were identified, and these grouped into two separate clusters with sequences from other parts of Africa and Asia. The T. parva p67 sequences were of the allele type 1, and parasites possessing this allele type are commonly associated with East Coast fever in eastern Africa. Analysis of the Ehrlichia and Anaplasma 16S rRNA gene sequences showed that they were closely related to Rickettsia africae and to a new Ehrlichia species variant recently found in China. Our R. africae 16S rRNA sequences grouped with R. africae isolates from Nigeria, Egypt and Benin. The information on tick species diversity and pathogens in the various tick species provides an indicator of potential transmission amongst cattle populations, and to humans, and can be useful to estimate disease risk and in control strategies.

Variant analysis of the sporozoite surface antigen gene reveals that asymptomatic cattle from wildlife-livestock interface areas in northern Tanzania harbour buffalo-derived T. parva.

Buffalo-derived Theileria parva can 'break through' the immunity induced by the infection and treatment vaccination method (ITM) in cattle. However, no such 'breakthroughs' have been reported in northern Tanzania where there has been long and widespread ITM use in pastoralist cattle, and the Cape buffalo (Syncerus caffer) is also present. We studied the exposure of vaccinated and unvaccinated cattle in northern Tanzania to buffalo-derived T. parva using p67 gene polymorphisms and compared this to its distribution in vaccinated cattle exposed to buffalo-derived T. parva in central Kenya, where vaccine 'breakthroughs' have been reported. Additionally, we analysed the CD8+ T cell target antigen Tp2 for positive selection. Our results showed that 10% of the p67 sequences from Tanzanian cattle (n = 39) had a buffalo type p67 (allele 4), an allele that is rare among East African isolates studied so far. The percentage of buffalo-derived p67 alleles observed in Kenyan cattle comprised 19% of the parasites (n = 36), with two different p67 alleles (2 and 3) of presumptive buffalo origin. The Tp2 protein was generally conserved with only three Tp2 variants from Tanzania (n = 33) and five from Kenya (n = 40). Two Tanzanian Tp2 variants and two Kenyan Tp2 variants were identical to variants present in the trivalent Muguga vaccine. Tp2 evolutionary analysis did not show evidence for positive selection within previously mapped epitope coding sites. The p67 data indicates that some ITM-vaccinated cattle are protected against disease induced by a buffalo-derived T. parva challenge in northern Tanzania and suggests that the parasite genotype may represent one factor explaining this.

Capture-based enrichment of Theileria parva DNA enables full genome assembly of first buffalo-derived strain and reveals exceptional intra-specific genetic diversity.

Theileria parva is an economically important, intracellular, tick-transmitted parasite of cattle. A live vaccine against the parasite is effective against challenge from cattle-transmissible T. parva but not against genotypes originating from the African Cape buffalo, a major wildlife reservoir, prompting the need to characterize genome-wide variation within and between cattle- and buffalo-associated T. parva populations. Here, we describe a capture-based target enrichment approach that enables, for the first time, de novo assembly of nearly complete T. parva genomes derived from infected host cell lines. This approach has exceptionally high specificity and sensitivity and is successful for both cattle- and buffalo-derived T. parva parasites. De novo genome assemblies generated for cattle genotypes differ from the reference by ~54K single nucleotide polymorphisms (SNPs) throughout the 8.31 Mb genome, an average of 6.5 SNPs/kb. We report the first buffalo-derived T. parva genome, which is ~20 kb larger than the genome from the reference, cattle-derived, Muguga strain, and contains 25 new potential genes. The average non-synonymous nucleotide diversity (πN) per gene, between buffalo-derived T. parva and the Muguga strain, was 1.3%. This remarkably high level of genetic divergence is supported by an average Wright's fixation index (FST), genome-wide, of 0.44, reflecting a degree of genetic differentiation between cattle- and buffalo-derived T. parva parasites more commonly seen between, rather than within, species. These findings present clear implications for vaccine development, further demonstrated by the ability to assemble nearly all known antigens in the buffalo-derived strain, which will be critical in design of next generation vaccines. The DNA capture approach used provides a clear advantage in specificity over alternative T. parva DNA enrichment methods used previously, such as those that utilize schizont purification, is less labor intensive, and enables in-depth comparative genomics in this apicomplexan parasite.

Ancient diversity and geographical sub-structuring in African buffalo Theileria parva populations revealed through metagenetic analysis of antigen-encoding loci.

An infection and treatment protocol involving infection with a mixture of three parasite isolates and simultaneous treatment with oxytetracycline is currently used to vaccinate cattle against Theileria parva. While vaccination results in high levels of protection in some regions, little or no protection is observed in areas where animals are challenged predominantly by parasites of buffalo origin. A previous study involving sequencing of two antigen-encoding genes from a series of parasite isolates indicated that this is associated with greater antigenic diversity in buffalo-derived T. parva. The current study set out to extend these analyses by applying high-throughput sequencing to ex vivo samples from naturally infected buffalo to determine the extent of diversity in a set of antigen-encoding genes. Samples from two populations of buffalo, one in Kenya and the other in South Africa, were examined to investigate the effect of geographical distance on the nature of sequence diversity. The results revealed a number of significant findings. First, there was a variable degree of nucleotide sequence diversity in all gene segments examined, with the percentage of polymorphic nucleotides ranging from 10% to 69%. Second, large numbers of allelic variants of each gene were found in individual animals, indicating multiple infection events. Third, despite the observed diversity in nucleotide sequences, several of the gene products had highly conserved amino acid sequences, and thus represent potential candidates for vaccine development. Fourth, although compelling evidence for population differentiation between the Kenyan and South African T. parva parasites was identified, analysis of molecular variance for each gene revealed that the majority of the underlying nucleotide sequence polymorphism was common to both areas, indicating that much of this aspect of genetic variation in the parasite population arose prior to geographic separation.

Limited genetic and antigenic diversity within parasite isolates used in a live vaccine against Theileria parva.

An infection and treatment protocol is used to vaccinate cattle against Theileria parva infection. Due to incomplete cross-protection between different parasite isolates, a mixture of three isolates, termed the Muguga cocktail, is used for vaccination. While vaccination of cattle in some regions provides high levels of protection, some animals are not protected against challenge with buffalo-derived T. parva. Knowledge of the genetic composition of the Muguga cocktail vaccine is required to understand how vaccination is able to protect against field challenge and to identify the potential limitations of the vaccine. The aim of the current study was to determine the extent of genetic and antigenic diversity within the parasite isolates that constitute the Muguga cocktail. High throughput multi-locus sequencing of antigen-encoding loci was performed in parallel with typing using a panel of micro- and mini-satellite loci. The former focused on genes encoding CD8(+) T cell antigens, believed to be relevant to protective immunity. The results demonstrate that each of the three component stocks of the cocktail contains limited parasite genotypic diversity, with single alleles detected at many gene/satellite loci and, moreover, that two of the components show a very high level of similarity. Thus, the vaccine incorporates very little of the genetic and antigenic diversity observed in field populations of T. parva. The presence of alleles at low frequency (<10%) within vaccine component populations also points to the possibility of variability in the content of vaccine doses and the potential for loss of allelic diversity during tick passage. The results demonstrate that there is scope to modify the content of the vaccine in order to enhance its diversity and thus its potential for providing broad protection. The ability to accurately quantify genetic diversity in vaccine component stocks will facilitate improved quality control procedures designed to ensure the long-term efficacy of the vaccine.

Molecular evolution of a central region containing B cell epitopes in the gene encoding the p67 sporozoite antigen within a field population of Theileria parva.

Protective immunity induced by the infective sporozoite stage of Theileria parva indicates a potential role for antibodies directed against conserved serologically reactive regions of the major sporozoite surface antigen p67 in vaccination to control the parasite. We have examined the allelic variation and determined the extent of B cell epitope polymorphism of the gene encoding p67 among field isolates originating from cattle exposed to infected ticks in the Marula area of the rift valley in central Kenya where the African cape buffalo (Syncerus caffer) and cattle co-graze. In the first of two closely juxtaposed epitope sequences in the central region of the p67 protein, an in-frame deletion of a seven-amino acid segment results in a truncation that was observed in parasites derived from cattle that co-grazed with buffalo. In contrast, the variation in the second epitope was primarily due to nonsynonymous substitutions, resulting in relatively low overall amino acid conservation in this segment of the protein. We also observed polymorphism in the region of the protein adjacent to the two defined epitopes, but this was not sufficient to provide statistically significant evidence for positive selection. The data indicates that B cell epitopes previously identified within the p67 gene are polymorphic within the Marula field isolates. Given the complete sequence identity of the p67 gene in all previously characterized T. parva isolates that are transmissible between cattle by ticks, the diversity observed in p67 from the Marula isolates in combination with the clinical reaction of the infected cattle is consistent with them originating from ticks that had acquired T. parva from buffalo.

Identification and sequence characterization of novel Theileria genotypes from the waterbuck (Kobus defassa) in a Theileria parva-endemic area in Kenya.

Waterbuck (Kobus defassa), an ungulate species endemic to the Eastern African savannah, is suspected of being a wildlife reservoir for tick-transmitted parasites infective to livestock. Waterbuck is infested by large numbers of Rhipicephalus appendiculatus, the tick vector for Theileria parva, and previous data suggests that the species may be a source of T. parva transmission to cattle. In the present study, a total of 86 cattle and 26 waterbuck blood samples were obtained from Marula, a site in Kenya endemic for East Coast fever (ECF) where the primary wildlife reservoir of T. parva the Cape buffalo (Syncerus caffer) is also common. To investigate for the presence of cattle-infective Theileria parasites, DNA specimens extracted from the blood samples were subjected to two diagnostic assays; a nested PCR based on the p104 gene that is specific for T. parva, and a reverse line blot (RLB) incorporating 13 oligonucleotide probes including all of the Theileria spp. so far described from livestock and wildlife in Kenya. Neither assay provided evidence of T. parva or Theileria sp. (buffalo) infection in the waterbuck DNA samples. By contrast, majority of the cattle samples (67.4%) were positive for T. parva using a nested PCR assay. The RLB assay, including a generic probe for the genus Theileria, indicated that 25/26 (96%) of the waterbuck samples were positive for Theileria, while none of the 11 Theileria species-specific probes hybridized with the waterbuck-derived PCR products. Phylogenetic analysis of 18S ribosomal RNA (18S rRNA) and internal transcribed spacer (ITS) sequences within the RLB-positive waterbuck samples revealed the occurrence of three Theileria genotypes of unknown identity designated A, B and C. Group A clustered with Theileria equi, a pathogenic Theileria species and a causative agent of equine piroplasmosis in domestic equids. However, DNA from this group failed to hybridize with the T. equi oligonucleotide present on the RLB filter probe, suggesting the occurrence of novel taxa in these animals. This was confirmed by DNA sequencing that revealed heterogeneity between the waterbuck isolates and previously reported T. equi genotypes. Group B parasites clustered closely with Theileria luwenshuni, a highly pathogenic parasite of sheep and goats reported from China. Group C was closely related to Theileria ovis, an apparently benign parasite of sheep. Together, these findings provided no evidence that waterbuck plays a role in the transmission of T. parva. However, novel Theileria genotypes detected in this bovid species may be of veterinary importance.

Geographic distribution of Theileria sp. (buffalo) and Theileria sp. (bougasvlei) in Cape buffalo (Syncerus caffer) in southern Africa: implications for speciation.

Strict control measures apply to movement of buffalo in South Africa including testing for Theileria parva, the causative agent of Corridor disease in cattle. The official test is a real-time hybridization PCR assay that amplifies the 18S rRNA V4 hyper-variable region of T. parva, T. sp. (buffalo) and T. sp. (bougasvlei). Mixed infections with the latter organisms affect diagnostic sensitivity due to PCR suppression. While the incidence of mixed infections in the Corridor disease endemic region of South Africa is significant, little information is available on the specific distribution and prevalence of T. sp. (buffalo) and T. sp. (bougasvlei). Specific real-time PCR assays were developed and a total of 1211 samples known to harbour these parasites were screened. Both parasites are widely distributed in southern Africa and the incidence of mixed infections with T. parva within the endemic region is similar (∼25-50%). However, a significant discrepancy exists in regard to mixed infections of T. sp. (buffalo) and T. sp. (bougasvlei) (∼10%). Evidence for speciation between T. sp. (buffalo) and T. sp. (bougasvlei) is supported by phylogenetic analysis of the COI gene, and their designation as different species. This suggests mutual exclusion of parasites and the possibility of hybrid sterility in cases of mixed infections.

Population genetic analysis and sub-structuring of Theileria parva in the northern and eastern parts of Zambia.

BACKGROUND: 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. METHODS: 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. 2.9.3.2, and LIAN computer softwares. RESULTS: 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. CONCLUSION: 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.

Analyses of genes encoding Theileria parva p104 and polymorphic immunodominant molecule (PIM) reveal evidence of the presence of cattle-type alleles in the South African T. parva population.

Restriction fragment length polymorphism analysis of PCR products (PCR-RFLP) and sequencing of the variable region of the p104 and PIM genes was performed on samples obtained from South African T. parva parasites originating from cattle on farms with suspected theileriosis and from buffalo. p104 and PIM PCR-RFLP profiles similar to those of the T. parva Muguga stock, an isolate that causes ECF in Kenya, were obtained from three of seven cattle samples collected on a farm near Ladysmith in KwaZulu-Natal Province. Amino acid sequences of the p104 and PIM genes from two of these samples were almost identical to the T. parva Muguga p104 and PIM sequences. This result supports findings from a recent p67 study in which p67 alleles similar to those of the T. parva Muguga stock were identified from the same samples. While these results suggest the presence of a cattle-derived T. parva parasite, reports of cattle-to-cattle transmission could not be substantiated and ECF was not diagnosed on this farm. Although extensive diversity of p104 and PIM gene sequences from South African T. parva isolates was demonstrated, no sequences identical to known cattle-type p104 and PIM alleles were identified from any of the buffalo T. parva samples analyzed. 'Mixed' PIM alleles containing both cattle- and buffalo-type amino acid motifs were identified for the first time, and there appeared to be selection of cattle-type and 'mixed'-type PIM sequences in the cattle samples examined.

Titrating Theileria parva: single stocks against combination of stocks.

Theileria parva is the causative agent of East Coast fever (ECF), an important cattle disease in East and Central Africa. One of the methods for control of ECF is 'infection and treatment', a procedure in which an animal is infected with the live parasite and at the same time treated with a long-acting oxytetracycline formulation, restraining the infection and allowing a protective cellular immune response to develop. Optimal immunizing doses were estimated using models of trichotomous response: dysimmunization (death or severe reaction during immunization), immunization failure (death or severe reaction during lethal challenge) and successful immunization (neither dysimmunization nor immunization failure). In this paper we present methods of interpreting immunization trials and apply these methods to previously unpublished data from two such trials: one with a mixture of three T. parva stocks and one with a single T. parva stock. We explain why titration trials conducted with a cocktail of antigens could predict a suboptimal immunization dose. Indeed it is possible for a combination of three individually efficient stocks to result in a mixture with which optimal immunization response might be difficult to achieve, because of averaging effects. The corresponding interpretation provides insights into why standard immunization trials for T. parva have not yielded the results that might be expected of them. The results of this work may also have implications for the use of antigen cocktails in cancer, HIV and malaria vaccine trials.

Epidemiological studies on tick-borne diseases of cattle in Central Equatoria State, Southern Sudan.

A herd-based study was carried out in Central Equatoria State, Southern Sudan, to study epidemiological aspects of tick-borne diseases. Six herds of cattle situated in three different locations were selected and investigated every 3 months during the year 2005. Blood smears for Giemsa staining and blood spots on filter paper for deoxyribonucleic acid extraction were collected from 600 apparently healthy indigenous cattle. A total of 69 (11.5%) samples showed the presence of piroplasms in Giemsa-stained blood smears, and polymerase chain reaction increased the detection limit to 297 (49.5%). Using reverse line blot, it was possible to detect and differentiate eight different piroplasms namely, Theileria parva (71.2%), Theileria mutans (73%), Theileria velifera (45.3%), Theileria taurotragi (2.7%), Theileria buffeli (0.5%), Theileria annulata (0.2%), Babesia bovis (1.7%), and Babesia bigemina (0.3%). Mixed infections were detected in 406 samples (67.7%) accounting for 17 different combinations. High infection of Theileria parva was reported among young calves compared to older cattle. The highest prevalence of Theileria parva was reported in the rainy season (October). The implications of these results on the epidemiology of tick-borne diseases are discussed with emphasis on East Coast fever.

Theileria parva ribosomal internal transcribed spacer sequences exhibit extensive polymorphism and mosaic evolution: application to the characterization of parasites from cattle and buffalo.

We sequenced the rRNA genes and internal transcribed spacers (ITS) of several Theileria parva isolates in an attempt to distinguish between the causative agents of East coast fever and Corridor disease. The small subunit (SSU) and large subunit (LSU) rRNA genes from a cloned T. p. lawrencei parasite were sequenced; the former was identical to that of T. p. parva Muguga, and there were minor heterogeneities in the latter. The 5.8S gene sequences of 11 T. parva isolates were identical, but major differences were found in the ITS. Six characterization oligonucleotides were designed to hybridize within the variable ITS1 region; 93.5% of T. p. parva isolates examined were detected by probe TPP1 and 81.8% of T. p. lawrencei isolates were detected by TPL2 and/or TPL3a. There was no absolute distinction between T. p. parva and T. p. lawrencei and the former hybridized with fewer of the probes than did the latter. It therefore seems that a relatively homogenous subpopulation of T. parva has been selected in cattle from a more diverse gene pool in buffalo. The ITSs of both T. p. parva and T. p. lawrencei contained different combinations of identifiable sequence segments, resulting in a mosaic of segments in any one isolate, suggesting that the two populations undergo genetic recombination and that their gene pools are not completely separate.

Characterization and discrimination of three Theileria parva stabilates involved in East Coast fever vaccination.

Three vaccine stabilates of Theileria parva, of which sporozoites are being used against East Coast fever, were characterized by immunological and molecular biology techniques before being used for a national vaccination campaign in Kenya. T. parva Marikebuni stabilates 316 and 3014, and T. parva Lanet were used in this study and were discriminated from other Kenyan field Theileria isolates. IFAT results showed that all the animals were producing antibodies regardless of the stock used. Primers designed on the TPR1 gene sequence were used for PCR and Decamers were used for RAPD. Specific DNA band patterns (1,877 bp; 1,059 bp, and 443 bp) for the three vaccine stocks were observed. These molecular markers could be used to trace vaccinated animals in Kenya and to identify which isolates are responsible for reactions in animals.

A PCR-based field evaluation of Theileria infections in cattle and ticks in Kenya.

Theileria parva is a hemoprotozoan parasite responsible for causing East Coast fever in east and central Africa. The vaccine currently available is an "infection and treatment" procedure which involves the injection of live sporozoites followed by drug therapy to prevent clinical illness. Before introducing potentially new strains of parasite into an area, however, it is crucial to check the disease situation in the field first. We looked at three different areas in Kenya: Limuru in which many cattle have already been vaccinated and Kitale and Kakamega which so far have not been vaccinated. Genus and species specific primers were used to test for the presence of Theileria species in blood and tick samples collected from the three areas. Limuru showed a cattle and tick infection prevalence of 27% and 2.3% respectively with T. parva. Kitale showed a cattle and tick infection prevalence of 100% and 14.2% respectively while Kakamega showed 100% and 0% respectively. Reasons for variations between areas involving vaccination status and epidemiological data are discussed.

Characterization of some Theileria parva stocks from Zambia using monoclonal antibodies.

Theileria parva parasites have been isolated from different location in Zambia where malignant theileriosis has been recorded. A total of 16 bovine lymphocytic cell lines infected with T. parva schizonts were characterized using a panel of anti-schizont monoclonal antibodies (MAbs). Comparison of the Theileria stocks isolated before (old) and after (new) the Muguga cocktail of T. parva from Kenya was used to vaccinate cattle against theileriosis in Zambia revealed differences in their reactivity against MAbs. The new isolates are showing MAb profiles similar to that exhibited by the Muguga cocktail which was used to vaccinate cattle in these areas between 1983 and 1989. These results suggest that the use of the Muguga cocktail to vaccinate animals against theileriosis in Zambia may have introduced Theileria stocks of different antigenic properties.

Use of the polymerase chain reaction for identification and quantification of Theileria parva protozoa in Rhipicephalus appendiculatus ticks.

The polymerase chain reaction (PCR) was adapted for detection of Theileria parva sporoblasts in Rhipicephalus appendiculatus ticks by comparison with staining of histological preparations of ticks with methyl green and pyronin (MGP). Two 32mer primers (IL174 and IL179) were used to amplify Theileria parva (Muguga isolate) DNA from the TPR 1 region of the genome by the PCR. Detection of T. parva was carried out with dissected salivary glands and whole ticks preserved in ethanol. Adult ticks which fed as nymphs on a T. parva infected calf were used in three experiments. Firstly, 70 whole ticks divided into 7 batches representing the rising and falling parasitaemia of the calf were used to show that detection of infection by the PCR was significantly correlated with MGP staining. Secondly, 120 dissected ticks were used from 4 different batches representative of the overall infection profile within the ticks to show a high correlation between PCR quantification within tick salivary glands and MGP count data of the paired gland. Thirdly, 120 ticks were used in batches selected for high and low infections. Bloodmeal contaminants from partially fed adult ticks, present in 60 out of the 120 ticks used, did not inhibit the PCR amplification of T. parva DNA. This experiment also showed a great increase in infection detection in partially fed batches of ticks compared to the untreated batches.

Theileria parva (Kasoba): isolation and challenge of cattle recovered from infection with other Theileria parva stocks.

A pathogenic Theileria stock was isolated from control cattle during an East Coast Fever (ECF) field immunization trial at Kasoba near Karonga town in northern Malawi. A stabilate of this stock caused severe fevers and prolonged parasitosis in Theileria parva naive cattle, resulting in the death of 5 out of 12 cattle despite treatment. In contrast, this parasite stock caused mild to moderate reactions in 17 cattle immunized with the trivalent T. parva stabilate except in 3 animals which developed severe reactions, and one of them died. Another time, cattle immunized with buffalo-derived Theileria parva (Serengeti transformed) resisted a potentially fatal challenge, with only mild to moderate reactions being recorded. The parasite stock was morphologically and serologically indistinguishable from Theileria parva (Muguga); it was virulent and could cause mortality, particularly in T. parva naive cattle. The parasite stock was designated Theileria parva (Kasoba).

Humoral immune responses to Theileria parva in cattle as measured by two-dimensional western blotting.

Humoral immune responses to schizont antigens from six stocks of Theileria parva were compared by two-dimensional Western blotting using sera from cattle that had been infected with a T. parva stock or a clone. Isoelectric points of a polymorphic immunodominant molecule (PIM) of schizonts that induces strong antibody responses in cattle ranged from acidic to basic. Molecular masses (Mr) of the PIM of the respective T. parva stocks were as follows: T. parva Muguga, 86 kDa; Mariakani, 83 kDa; Marikebuni, 83 kDa; Uganda, 83 kDa; T. parva Boleni, 83 kDa; and T. parva 7014, 100 kDa. Among nine cattle infected with T. parva Muguga, four produced antibodies to a basic antigen having an Mr of 32 kDa. The PIM of T. parva Muguga, T. parva Boleni, and T. parva 7014 reacted strongly with serum obtained from an animal that had been infected with T. parva Muguga. Two-dimensional Western blotting using antischizont monoclonal antibodies enabled us to differentiate between stocks of T. parva.