First report of Theileria annulata in Nigeria: Findings from cattle ticks in Zamfara and Sokoto States.

BACKGROUND: Ticks and tick-borne pathogens (TBPs) represent a significant economic burden to cattle farming in sub-Saharan Africa including Nigeria. However, in the northern part of this country, where the largest livestock population resides, little is known about the contemporary diversity of ticks and TBPs. This area is particularly vulnerable to climate change, undergoing marked transformation of habitat and associated flora and fauna that is also likely to include ticks. This study aimed to document the occurrence of tick species and Apicomplexan TBPs in cattle from north-western Nigeria. METHODS: In 2017, ticks were collected from cattle in Zamfara and Sokoto States and identified morphologically. Additionally, a subset of ticks was screened molecularly for the detection of apicomplexan DNA. RESULTS: A total of 494 adult ticks were collected from 80 cattle in Zamfara and 65 cattle in Sokoto State. Nine tick species were encountered, among which the presence of one, Hyalomma turanicum, had not previously been recorded in Nigeria. Hyalomma rufipes was the most prevalent tick infesting cattle in Zamfara State (76%), while Hyalomma dromedarii was the most prevalent in Sokoto State (44%), confirming the widespread transfer of this species from camels onto livestock and its adaptation to cattle in the region. Of 159 ticks screened, 2 out of 54 (3.7%) from Zamfara State and 29 out of 105 (27.6%) from Sokoto State harboured DNA of Theileria annulata, the agent of tropical theileriosis. CONCLUSIONS: This study confirms the presence of a broad diversity of tick species in cattle from north-western Nigeria, providing the first locality records for Zamfara State. The occurrence of H. turanicum indicates a distribution of this tick beyond northern Africa. This study provides the first report for T. annulata in Nigerian ticks. Given its enormous burden on livestock farming in north Africa and across Asia, further investigations are needed to better understand its epidemiology, vector transmission and potential clinical significance in cattle from northern Nigeria and neighbouring Sahelian countries.

Coexistence of Multiple Theileria annulata Genotypes Circulating in Neonatal Calves in Semi-arid India.

BACKGROUND: Knowledge of local isolates and strains is a prerequisite for the development of either effective mass vaccination strategy or a suitable molecular marker-based diagnostic tool. PURPOSE: The pathogenesis of Bovine tropical theileriosis (BTT), caused by Theileria annulata in susceptible ruminants, is known to vary depending upon the nature of isolate and strain involved. Therefore, RFLP and sequencing-based characterization of Indian isolates of T. annulata were attempted using TAMS gene. METHOD: In the present study, TAMS 1 gene of T. annulata was amplified from 25 naturally infected calves from the BTT endemic semi-arid zone of Northern India. The amplified products were then digested with three restrictions enzymes viz., Taq I, Rsa I, and Alu I to find out the variations in pattern of restriction digests, so as to have an idea about the various isolates of T. annulata present in the studied area. Around 14 samples covering all the variants (from the PCR-RFLP patterns) were sequenced and submitted in NCBI (MH277607-MH277620). RESULT: Coexistence of 4 variant genotypes was detected upon in-silico analysis of RFLP and sequence variations. CONCLUSION: The nucleotide variations alongside the chromatogram analysis revealed point mutations leading to presence of noticeable genetic diversity among the isolates.

Molecular Detection, Phylogenetic Analysis, and Genetic Diversity of Theileria annulata, Babesia bigemina, and Anaplasma marginale in Cattle in Three Districts of Egypt.

BACKGROUND: Under the poor hygienic conditions, tick-borne pathogens cause severe economic losses to the cattle industry. PURPOSE: The current study investigated the presence of Theileria annulata, Babesia bigemina, and Anaplasma marginale, the most relevant tick-borne pathogens in cattle, in 3 provinces of Egypt utilizing species-specific PCR assays. METHODS: PCR was conducted, on bovine blood specimens, using primers targeting the T. annulata merozoite-piroplasm surface antigen (Tams1, 768 bp), A. marginale major surface protein-1b gene (msp1b, 265 bp), and B. bigemina small subunit ribosomal RNA gene (SSrRNA, 543 bp). RESULTS: PCR findings revealed overall prevalences of T. annulata, B. bigemina, and A. marginale as 22.0% (33/150), 19.33% (29/150), and 10.6% (16/150), respectively. The co-infection with two or three pathogens was detected in 20.0% (30/150) of examined specimens. Sequence analyses indicated that T. annulata and A. marginale varied from those of corresponding GenBank sequences revealing percent identities ranging from 90.68 to 97.75% and from 94.98 to 98.63%, respectively. On the other hand, the obtained B. bigemina sequences showed a high similarity with those previously reported in GenBank with a percent identity ranging from 98.85 to 100%. CONCLUSION: T. annulata was the most prevalent tick-borne pathogen in examined bovine specimens. The genetic diversity of markers used for identification of T. annulata and A. marginale should be highly considered.

Discovery of a new Theileria sp. closely related to Theileria annulata in cattle from Sri Lanka.

Theileria annulata is a haemoprotozoan parasite that causes a cancer-like illness known as tropical theileriosis in cattle. In the course of analyzing the genetic diversity of T. annulata in Sri Lanka, we observed that merozoite-piroplasm surface antigen (tams1) and surface protein (tasp)-like gene sequences obtained from bovine blood DNA samples, which were PCR-positive for T. annulata, were conserved but shared low identity with T. annulata GenBank sequences. Moreover, the 18S rRNA sequences from the Sri Lankan samples contained ten unique single-nucleotide polymorphisms compared with all known T. annulata sequences. The cytochrome b (cob) gene sequences isolated from the Sri Lankan samples were highly conserved and shared low identity scores with similarly conserved T. annulata sequences from GenBank. Phylogenetic analysis showed that the Sri Lankan tams1-like, tasp-like, 18S rRNA, and cob sequences clustered together and formed sister clades to the common ancestors of all known T. annulata and Theileria lestoquardi sequences. These findings demonstrated that the Sri Lankan cattle were not infected with T. annulata but with a new Theileria sp. (designated as Theileria sp. Yokoyama) closely related to T. annulata.

Selection of Genetic Markers to Determine Diversity in Theileria annulata Populations after Recombination.

OBJECTIVE: Selecting polymorphic mini- and microsatellite markers to determine genetic diversity and chromosomal regions exhibiting elevated rates of recombination in Theileria annulata populations after recombination. METHODS: The Unipro UGENE software was used to select markers. A score at which 10 times more tandem repeats (TRs) were identified in the real DNA sequence than those in the scrambled sequences of T. annulata was used as the cutoff. TRs containing minimum three nucleotides in length for microsatellite and six nucleotides for minisatellite regions and having a repeat motif identity between 96%-100% with the unit size repeated minimum three times were screened through the whole genome using the suffix array algorithm. RESULTS: A total of 359 minisatellites and 8973 microsatellites were identified. TRs were screened one by one through the whole genome; mini- and microsatellites representing a single region and having suitable regions for primer design were selected based on their localization on T. annulata chromosomes, their repeat motif identity (>96%), and their repeat length (<1500 bp). The primers used to amplify selected candidates were designed, and each primer was used to check 27 different isolates of T. annulata. CONCLUSION: In the present study, a total of 13 polymorphic mini- and microsatellite markers located on the different chromosomes were selected to determine the population diversity of T. annulata.

Identification of 12 Piroplasms Infecting Ten Tick Species in China Using Reverse Line Blot Hybridization.

Piroplasmosis, a disease of domestic and wild animals, is caused by tick-borne protozoa in the genera of Theileria and Babesia. There is limited information available about the prevalence of piroplasmosis in ticks in China, and to assess the potential threat of piroplasmosis in China, we investigated the infections of ovine and bovine Babesia and Theileria species in ticks collected from cattle, yaks, sheep, horses, and camels in several regions of China where tick-borne diseases have been reported. In total, 652 ticks were collected from the animals in 6 provinces of China. Babesia spp. and Theileria spp. were detected with a PCR-RLB method and identified by sequencing. Overall, 157 ticks (24.1%) were infected with 5 Babesia and 4 Theileria species. Among tested tick samples, 134 (20.6%) were single infections with 1 of 7 piroplasm species, with Theileria annulata (118/652, 18.1%) being dominant. Only 23 (3.5%) tick samples were double or triple infected, Theileria luwenshuni and Theileria sinensis (18/652, 2.8%) were frequently observed in co-infections. Some piroplasm species were carried by ticks that were not previously reported to be vectors.

Molecular and Parasitological Survey of Ovine Piroplasmosis, Including the First Report of Theileria annulata (Apicomplexa: Theileridae) in Sheep and Goats from Turkey.

Blood and tick samples were collected from 333 apparently healthy sheep and 257 goats as well as 10 sheep exhibiting clinical signs of babesiosis in Adana, Gaziantep, and Adiyaman Provinces in southern Turkey. Fully engorged female ticks were selected and maintained in an incubator until they oviposited. The tick carcasses and their egg masses were examined. Piroplasms compatible with Babesia spp. and Theileria spp. were observed in both symptomatic and asymptomatic small ruminants. Genomic DNA isolates from blood of ovine, tick samples, and egg masses were screened for piroplasms by utilizing 18S rRNA polymerase chain reaction and reverse line blotting (RLB) assays. Parasitemia ranged from 0.01% to 5.6% of erythrocytes in clinical cases. RLB showed positivity in 239 (40.5%) of the sampled apparently healthy sheep and goats and revealed the presence of three Theileria and one Babesia species. Theileria ovis was the most prevalent (35.4%), followed by Babesia ovis (5.4%), Theileria annulata (3.9%), and Theileria sp. MK (0.3%). Thirty-two small ruminants infected with T. ovis were also infected with B. ovis One animal infected with T. ovis was also infected with Theileria sp. MK. Ticks were identified as Rhipicephalus bursa, Rhipicephalus turanicus, Hyalomma excavatum, Haemaphysalis parva, and Hyalomma anatolicum Egg masses of two female R. bursa carcasses were infected with B. ovis This is the first report of theileriosis caused by T. annulata in sheep and goats in Turkey.

Theileria lestoquardi displays reduced genetic diversity relative to sympatric Theileria annulata in Oman.

The Apicomplexan parasites, Theileria lestoquardi and Theileria annulata, the causative agents of theileriosis in small and large ruminants, are widespread in Oman, in areas where cattle, sheep and goats co-graze. Genetic analysis can provide insight into the dynamics of the parasite and the evolutionary relationship between species. Here we identified ten genetic markers (micro- and mini-satellites) spread across the T. lestoquardi genome, and confirmed their species specificity. We then genotyped T. lestoquardi in different regions in Oman. The genetic structures of T. lestoquardi populations were then compared with previously published data, for comparable panels of markers, for sympatric T. annulata isolates. In addition, we examined two antigen genes in T. annulata (Tams1 and Ta9) and their orthologues in T. lestoquardi (Tlms1 and Tl9). The genetic diversity and multiplicity of infection (MOI) were lower in T. lestoquardi (He=0.64-0.77) than T. annulata (He=0.83-0.85) in all populations. Very limited genetic differentiation was found among T. lestoquardi and T. annulata populations. In contrast, limited but significant linkage disequilibrium was observed within regional populations of each species. We identified eight T. annulata isolates in small ruminants; the diversity and MOI were lower among ovine/caprine compared to bovine. Sequence diversity of the antigen genes, Tams1 and Ta9 in T. annulata (π=0.0733 and π=0.155 respectively), was 10-fold and 3-fold higher than the orthologous Tlms1 and Tl9 in T. lestoquardi (π=0.006 and π=0.055, respectively). Despite a comparably high prevalence, T. lestoquardi has lower genetic diversity compared to sympatric T. annulata populations. There was no evidence of differentiation among populations of either species. In comparison to T. lestoquardi, T. annulata has a larger effective population size. While genetic exchange and recombination occur in both parasite species, the extent of diversity, overall, is less for T. lestoquardi. It is, therefore, likely that T. lestoquardi evolved from an ancestor of present day T. annulata and that this occurred either once or on a limited number of occasions.

Population diversity of Theileria annulata in Portugal.

The tick-borne protozoan parasite Theileria annulata causes tropical theileriosis, a severe disease of cattle that occurs across the Mediterranean littoral, the Middle East and Southern Asia. In the Mediterranean region, the disease has long been perceived as being a constraint to livestock production in North Africa and Turkey but was believed to have minimal impact in Southern European countries. It has recently been demonstrated that in Southern Portugal the prevalence of T. annulata is approximately 30%. While the population genetics of the parasite and the multiplicity of infection in the bovine host have been studied in a number of countries, no information is currently available on the composition of the parasite population in Southern Europe or its relationship to populations in bordering regions. A parasite genotyping system, based on micro- and mini-satellite amplification, was used to perform genetic analysis of T. annulata populations from T. annulata infected cattle in twelve farms in Southern Portugal. A diversity of genotypes and a high multiplicity of infection were found, suggesting that the parasite possesses a panmictic population in this region. In comparison with genotypes found in Tunisia and Turkey, parasites from Portugal form a genetically distinct group and show lower genetic diversity.

Identification of piroplasm infection in questing ticks by RLB: a broad range extension of tick-borne piroplasm in China?

Sensitive and specific diagnostic method for rapid and simultaneous detection and discrimination of the different species is needed for an effective control of piroplasmosis. Here, a reverse line blot (RLB) assay was developed for piroplasm detection. A general pair of primer based on 18S ribosomal RNA (rRNA) gene was used to amplify V4 region of 18S rRNA gene. General and specific probes for 13 piroplasm species were cited from previous publications or designed according to the alignment of 18S rRNA gene sequences. For sensitivity test of RLB assay, serially diluted plasmids of the different species were used to access the sensitivity of the RLB. Four hundred and fifty tick samples collected from grass from different provinces of China were then detected. The result indicated that the RLB assay is highly specific and sensitive, detecting up to 10(2) copies/µl of recombinant plasmid DNA. Multiple piroplasms were detected as single or mixed infection from tick species. Eight piroplasm species, most of which were Theileria annulata (33/450, 7.3 %) or Babesia sp. Xinjiang (30/450, 6.7 %), were found to infect with 89 tick samples in four tick species; no infections with Babesia major, Babesia ovata, Babesia bigemina, Theileria sergenti, or Theileria equi were detected. The piroplasms species-specific RLB assay may have potential clinical application in the simultaneous detection and differentiation of Babesia and Theileria species.

Molecular and Phylogenetic analysis revealed new genotypes of Theileria annulata parasites from India.

BACKGROUND: Tick borne diseases impinge cattle worldwide causing mortality and resulting in huge economic losses. Theileriosis is one of the important tick borne diseases mainly caused by Theileria annulata and one of the commonly occurring infections among the livestock. T. annulata causes immense loss to the livestock industry and therefore, efficacious eradication and control strategies are needed for the control of the disease. Genetic diversity among T. annulata parasites is another important aspect which is overlooked in India. Thus, the present study aims to evaluate the prevalence along with genetic diversity and phylogeny of the prevailing T. annulata population of India. METHODS: Genomic DNA was extracted from cattle blood samples (n = 862) from different regions of Andhra Pradesh. Molecular diagnosis using T. annulata 18S rRNA based PCR was performed to detect parasites in cattle. Further, 18S rRNA gene was cloned and sequenced to determine similarity and diversity from the known T. annulata sequences. RESULTS: We observed an overall prevalence rate of 32.40 % T. annulata infection in Andhra Pradesh based on PCR assay. The sequence analysis revealed novel genotypes among the T. annulata strains from India. Thirteen strains showed closed proximity with a strain from China whereas one Indian strain showed similarity with a South African strain [Theileria sp (buffalo)] based on phylogenetic analysis. Nucleotide heterogeneity of the 18S rRNA sequence among the strains examined varied from 0.1 to 8.6 % when compared with the published strains. CONCLUSION: The present study provides us with the molecular prevalence of theileriosis, and will support the accomplishment of actions or in design of strategy to control theileriosis transmission to cattle. Additionally, it highlights the emergence of strains with novel genotypes from India.

Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle.

Aim to construct a simple and efficient diagnostic assay for Theileria annulata and Theileria sergenti, a multiplex polymerase chain reaction (PCR) method was developed in this study. Following the alignment of the related sequences, two primer sets were designed specific targeting on T. annulata cytochrome b (COB) gene and T. sergenti internal transcribed spacer (ITS) sequences. It was found that the designed primers could react in one PCR system and generating amplifications of 818 and 393 base pair for T. sergenti and T. annulata, respectively. The standard genomic DNA of both species Theileria was serial tenfold diluted for testing the sensitivity, while specificity test confirmed both primer sets have no cross-reaction with other Theileria and Babesia species. In addition, 378 field samples were used for evaluation of the utility of the multiplex PCR assay for detection of the pathogens infection. The detection results were compared with the other two published PCR methods which targeting on T. annulata COB gene and T. sergenti major piroplasm surface protein (MPSP) gene, respectively. The developed multiplex PCR assay has similar efficient detection with COB and MPSP PCR, which indicates this multiplex PCR may be a valuable assay for the epidemiological studies for T. annulata and T. sergenti.

An epidemiological survey of bovine Babesia and Theileria parasites in cattle, buffaloes, and sheep in Egypt.

Cattle, buffaloes, and sheep are the main sources of meat and milk in Egypt, but their productivity is thought to be greatly reduced by hemoprotozoan parasitic diseases. In this study, we analyzed the infection rates of Babesia bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis, using parasite-specific PCR assays in blood-DNA samples sourced from cattle (n=439), buffaloes (n=50), and sheep (n=105) reared in Menoufia, Behera, Giza, and Sohag provinces of Egypt. In cattle, the positive rates of B. bovis, B. bigemina, T. annulata, and T. orientalis were 3.18%, 7.97%, 9.56%, and 0.68%, respectively. On the other hand, B. bovis and T. orientalis were the only parasites detected in buffaloes and each of these parasites was only found in two individual DNA samples (both 2%), while one (0.95%) and two (1.90%) of the sheep samples were positive for B. bovis and B. bigemina, respectively. Sequence analysis showed that the B. bovis Rhoptry Associated Protein-1 and the B. bigemina Apical Membrane Antigen-1 genes were highly conserved among the samples, with 99.3-100% and 95.3-100% sequence identity values, respectively. In contrast, the Egyptian T. annulata merozoite surface antigen-1 gene sequences were relatively diverse (87.8-100% identity values), dispersing themselves across several clades in the phylogenetic tree containing sequences from other countries. Additionally, the T. orientalis Major Piroplasm Surface Protein (MPSP) gene sequences were classified as types 1 and 2. This is the first report of T. orientalis in Egypt, and of type 2 MPSP in buffaloes. Detection of MPSP type 2, which is considered a relatively virulent genotype, suggests that T. orientalis infection may have veterinary and economic significance in Egypt. In conclusion, the present study, which analyzed multiple species of Babesia and Theileria parasites in different livestock animals, may shed an additional light on the epidemiology of hemoprotozoan parasites in Egypt.

Phylogenetic analysis of bovine Theileria spp. isolated in south India.

The objective of the present study is to determine the phylogenetic position of the Theileria organisms in blood of cattle of southern India using molecular tools. Theileria annulata (Namakkal isolate, Tamil Nadu) and three Theileria field isolates (free of T. annulata) from Wayanad, Kerala (Wayanad 1, 2, 3) were used. The small subunit ribosomal RNA (SSU rRNA) and major piroplasm surface protein (MPSP) gene products were cloned, sequenced and the phylogenetic tree constructed. SSU rRNA gene of Wayanad 1 isolate (JQ706077) revealed maximum identity with Theileria velifera or Theileria cervi. The phylogenetic tree constructed based on SSU rRNA genes revealed that Wayanad 1 isolate belonged to a new type which share common ancestor with all the other theilerial species while Wayanad 2 and 3 isolates (JX294459, JX294460) were close to types A and C respectively. Based on MPSP gene sequences, Wayanad 2 and 3 (JQ706078, JX648208) isolates belonged to Type 1 and 3 (Chitose) respectively. When, the previously reported MPSP type 7 is also considered from the same study area, Theileria orientalis types 1, 3 and 7 are observed in south India. SSU rRNA sequence of South Indian T. annulata (JX294461) showed a maximum identity with Asian isolates while the Tams1 merozoite surface antigen (MSA) gene (JX648210) showed maximum identity with north Indian isolate.

Molecular epidemiology of Theileria annulata and identification of 18S rRNA gene and ITS regions sequences variants in apparently healthy buffaloes and cattle in Pakistan.

A molecular epidemiological survey was conducted to determine the prevalence of piroplasms in buffaloes and cattle from Sheikhupura and Okara districts of Punjab, Pakistan using reverse line blot (RLB) hybridization assay. The genetic diversity within 18S rRNA gene and ITS regions sequences of various obtained Theileria species (spp.) was also investigated. Briefly, 102 blood samples from buffaloes and cattle in the study districts were collected on blood collection cards and brought to the laboratory. DNA was extracted; the V4 hypervariable region of 18S rRNA was amplified and analyzed using RLB. Out of total samples analyzed, 61 (59.8%) were hybridized with Babesia/Theileria (B/T) genus-specific probe. Only one species of piroplasm was detected in buffaloes and cattle in study districts, i.e. Theileria (T.) annulata. Six samples only hybridized with B/T genus-specific and Theileria genus-specific probes but not with any species-specific probe indicating the presence of novel species or variants. The sequences of 18S rRNA gene and ITS regions of these six samples revealed the presence of T. annulata variants as confirmed through sequence identity estimation and phylogenetic analyses. Meanwhile, an unexpected sequence variation was observed within the 18S rRNA gene and ITS regions sequences of T. annulata identified in the present study. This is the first report on the simultaneous detection of species of piroplasms infecting buffaloes and cattle in Pakistan and molecular characterization of T. annulata 18S rRNA gene and ITS regions. The present study may address the new insights into the epidemiology of theileriosis which will help researches in designing control strategies and developing various molecular diagnostic tools at national level.

Population diversity and multiplicity of infection in Theileria annulata.

The tick-borne apicomplexan parasite Theileria annulata is endemic in many sub-tropical countries and causes the bovine disease tropical theileriosis. Although the parasite is known to be highly diverse, detailed information is lacking on the genetic structure of natural populations and levels of multiplicity of infection in the cattle host. With the widespread deployment of live attenuated vaccines and the emergence of drug-resistant parasites in the field, it is vital to appreciate the factors which shape genetic diversity of the parasite both within individual hosts and in the wider population. This study addresses these issues and represents an extensive genetic analysis of T. annulata populations in two endemic countries utilising a high-throughput adaptation of a micro- and mini-satellite genotyping system. Parasite material was collected from infected cattle in defined regions of Turkey and Tunisia to allow a variety of analyses to be conducted. All animals (n=305) were found to harbour multiple parasite genotypes and only two isolates shared an identical predominant multi-locus profile. A modelling approach was used to demonstrate that host age, location and vaccination status play a measurable role in determining multiplicity of infection in an individual animal. Age was shown to positively correlate with multiplicity of infection and while positive vaccination status exerted a similar effect, it was shown to be due not simply to the presence of the immunising genotype. Importantly, no direct evidence was found for the immunising genotype spreading or recombining within the local parasite community. Genetic analysis confirmed the tentative conclusion of a previous study that the parasite population appears to be, in general, panmictic. Nevertheless, evidence supporting linkage disequilibrium and a departure from panmixia was uncovered in some localities and a number of explanations for these findings are advanced.

Sequence analysis of Theileria annulata surface protein in Chinese isolates.

OBJECTIVE: To study the TaSP polymorphism in three Chinese isolates of Theileria annulata. METHODS: The isolates from Inner Mongolia Autonomous Region, Ningxia Hui Autonomous Region and Xinjiang Uygur Autonomous Region were cultured in RPMI 1640 medium. TaSP gene was amplified from genomic DNA extracted from schizonts using polymerase chain reaction (PCR) and sequenced. Its amino acid sequence comparison was carried out with Clustal W2 multiple sequence alignment program. Molecular component and motif prediction were performed with online servers. RESULTS: The comparison of TaSP amino acid sequences of the three isolates showed that the central region (aa position 38-161) predicted to be the highly immunogenetic domain was polymorphic both in size and amino acid sequence, while the N-terminal (first 37 aa) and C-terminal (last 154 aa) parts were strongly conserved. Phylogenetic analysis and percentage identity revealed that the Chinese isolates were closely related to the isolates from Turkey, but quite different from those of India, Morocco and Tunisia. More importantly, variability was noticed among Chinese isolates, which caused both the location and number's differences of motif (casein kinase II phosphorylation sites) among three TaSP sequences. CONCLUSION: TaSP polymorphism exists in the Chinese isolates of T. annulata.

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.

Discrimination between Theileria lestoquardi and Theileria annulata in their vectors and hosts by RFLP based on the 18S rRNA gene.

Theileria lestoquardi and T. annulata can occur in similar vectors, and current available probes based on the 18S rRNA gene showed cross-reaction between the two species. However, we developed a species-specific RFLP test based on the MspI restriction enzyme, able to cut amplified products from T. lestoquardi only and to discriminate the two species in both tick and blood samples.

Reverse line blot hybridization used to identify hemoprotozoa in Minorcan cattle.

Piroplasmosis, a tick-borne protozoal disease, is an important disease affecting domestic and wild animals. We performed PCR-based reverse line blot hybridization (RLB) assays on blood samples obtained from 133 cattle exposed to ticks in field conditions in Minorca (Balearic Islands, Spain) in three different seasons. The oligonucleotides used were those for Theileria annulata, T. buffeli, T. taurotragi, T. velifera, Babesia bigemina, B. bovis, B. divergens, and B. major. The RLB technique allowed the simultaneous identification of T. annulata, T. buffeli, B. bigemina, and B. bovis as the piroplasms present in cattle in Minorca. Of the 133 animals, only 4 were not infected by any of the studied parasites. The results indicated endemic piroplasm infection in cattle in Minorca; especially important was the presence of T. annulata. The RLB was highly sensitive and allowed the simultaneous detection and identification of the Theileria and Babesia species in carrier cattle, which cannot be achieved by classical identification methods.